JP2022137048A - Automated tissue engineering module - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for the automated culture, proliferation, differentiation, production and maintenance of tissue engineered products.

SOLUTION: A automated method for cell culture including cells of an autologous, allogenic, or xenogenic origin, includes (a) loading a cell into a bioreactor fluid-connected with a media reservoir and flow system, the bioreactor having one or more sensors for positively monitoring and adjusting by a microprocessor during cell culture; (b) proliferating and/or differentiating the cells within the bioreactor; and (c) using the microprocessor, during the proliferating and/or differentiating, to positively monitor and automatically adjust parameters such as the temperature of cell culture, a pH, dissolved gas, metabolic turnover, and an image, indirectly evaluating the status of the cell proliferation as a function of time by the detection of metabolic turnover, and determining the level of confluence by sensor-based monitoring and evaluating on the status of the cell proliferation.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

The present invention relates to devices, methods and systems for automated culture, expansion, differentiation, generation and maintenance of tissue engineered products. Such systems, methods and products find use in a variety of clinical and laboratory settings.

Throughout this application, various references are cited within parentheses to more fully describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the disclosure of the present application.

During the past few years, researchers have developed and used different cell culture and tissue engineering techniques for the culture and production of various types of cell implants. Such systems are described, for example, in US Pat. Nos. 5,041,138, 5,842,477, 5,88
2,929, 5,891,455, 5,902,741, 5,994
, 129, 6,048,721 and 6,228,635.
Bioreactor systems have also been developed for the culture of cells and cell implants and are disclosed, for example, in US Pat. Nos. 5,688,687, 5,728,581,
Nos. 5,827,729 and 6,121,042.

The foregoing methods and systems generally employ conventional experimental culture techniques using standard culture equipment for cell seeding of selected cell populations onto scaffolds. Thus, the implants produced simply contain an expanded population of cells growing on some type of biopolymer support, for which any manipulation of the cellular environment is , the endogenous cellular production of cytokines present in any standard cell culture, and the application of shear and/or physical stress due to circulation of the cell culture medium, and the physical nature of the support on which the cells are seeded. Limited to operations. These systems neither address nor are capable of producing tissue implants containing proliferated and differentiated cells representative of tissue developing in vivo, and can be successfully incorporated in vivo. Nor is it further embedded within the selected scaffold. Additionally, known methods and systems provide multifunctional performance of all steps of biopsy tissue digestion to yield dissociated cells, subsequent cell seeding onto growth substrates, within a single automated tissue engineering system.
Inability to generate cell number expansion, controlled differentiation, tissue formation, and tissue implants. A major reason for this is that known culture systems are capable of automatically assessing and manipulating the changing environment around a developing implant so that cells progressively proliferate and differentiate into the desired implant. This is because it is not advanced in that it cannot be done.

In addition, conventional culture methods and systems are labor intensive, suffer from contamination, and have variable degrees of culture success due to human error and lack of continuous performance assessment. Conventional culture systems require that most of the initial steps in preparing cells for seeding (i.e., tissue digestion, cell selection) be performed manually, which is time consuming and productive. The quality of the tissue produced is unreliable and problems of culture contamination are prone. This system suffers from inherent design limitations that limit cell and tissue culture processes, the inability to sufficiently monitor and modify the environment to support tissue development, and the inability to implement effective quality control measures. Due to the lack of available techniques, the automated preparation of tissue engineered implants from primary or progenitor cells cannot be supported.

Accordingly, there is a real and unmet need for improved systems of in vitro and ex vivo tissue engineering that can consistently meet the operational requirements associated with different steps in the development and production of tissue engineered implants. still exists. Of particular importance is the ability to create functional tissue constructs in which:
The cells present are active, differentiated and already express extracellular matrix. This means more than, and is markedly different from, simple stimulation of the mature in vivo environment that exists at the host site. The reason for this is the functional de novo
) because tissue preparation essentially requires cells to progress through a series of developmental stages as part of an ex vivo procedure.

To address both clinical and research requirements, new devices, methods and systems are being developed that obviate some of the disadvantages and limitations of conventional ex vivo culture techniques and systems.

The present invention relates to user-friendly automated systems for cell culture and tissue engineering that can be used in a variety of clinical and research settings for both human and veterinary applications. .

As used herein, "tissue engineering"
'can be defined as 'the application of engineering and life science principles and methods toward the fundamental understanding and development of biological alternatives to repair, maintain and improve tissue function'. This definition is that a biological substitute is a cell or combination of different cells that can be implanted onto a substrate or scaffold formed of a biocompatible material to form tissue, in particular an implantable tissue construct. It is intended to encompass procedures. Furthermore, note that the cells involved in the tissue engineering process can be autologous, allogeneic, or xenogeneic.

The tissue engineering system of the present invention is designed to perform all activities under sterile operating conditions. The system is fully automated, portable, multi-functional in operation, and performs/provides one or more of the following.
Aseptic receipt/storage of tissue biopsies,
automatic monitoring of the digestive process,
Digestion of biopsy tissue to obtain dissociated cells,
cell sorting and selection, including safe waste collection;
cell seeding onto or into a growth substrate or scaffold;
proliferation of cells to increase the cell population;
washing the cells and harvesting the cells,
seeding cells onto or into tissue engineering scaffolds or matrices;
cell differentiation, which allows for the specialization of cell activity;
tissue formation,
mechanical and/or biochemical stimulation to promote tissue maturation;
Collection of tissue engineered constructs/implants for reconstructive surgery and storage and transport of implantable tissue.

The tissue engineering system of the present invention is preprogrammed to perform each of the above steps individually, sequentially, or in a specific predetermined partial order, as desired and necessary. It is possible. Further, each of these steps, or any combination thereof, is accomplished within one or more bioreactors on the tissue engineering module. In operation, this tissue engineering system is pre-programmed and automatically controlled, thus requiring minimal user intervention, resulting in minimal risk of contamination.
Enhances the efficacy and reproducibility of cell culture and/or tissue engineering processes. The tissue engineering system and components thereof of the present invention are operable under conditions of microgravity and/or weightlessness, where such systems and components are used for space research.

The system of the present invention allows primary or progenitor cells to further grow tissue implants,
Designed to be isolated from donor tissue for differentiation and production. Alternatively, cell lines may also be used either alone or in combination with other cell sources.

According to the present invention, an automated tissue engineering system that supports physiological cellular functions and at least one bioreactor that facilitates the production of tissue constructs from cell and tissue sources. A housing is provided. The housing also supports a liquid containment system in fluid communication with the bioreactor. Associated with the housing and/or the bioreactor is provided in the liquid containment system,
Sensors that monitor physiological parameters of fluids. A microprocessor located within this housing is coupled to the bioreactor and the liquid containment system and functions to control their functions. The microprocessor can also independently control environmental conditions within the system.

According to another aspect of the present invention, a system for cell and tissue engineering is provided, comprising a portable, sterile tissue engineering module that performs tissue digestion, cell seeding onto growth substrates, Having one or more bioreactors to base tissue formation with cell growth, cell seeding onto differentiation scaffolds, cell differentiation, and subsequent maturation into functional tissue for transplantation. The bioreactor is operably connected in a non-perfusion fashion with media flow and reservoir systems for delivery of reagents and collection of waste liquids. The bioreactor and/or medium flow system optionally includes a gas exchange component, which utilizes a semi-permeable membrane to allow gas movement, thereby Control the level of dissolved gases. This tissue engineering module operatively interacts with a central microprocessor controlled base unit that automatically monitors the progress of the cell culture or tissue engineering process to achieve different stages of cell culture and tissue development within this bioreactor. Adjust the environmental conditions to meet the requirements of Deviations from ideal conditions are detected by various sensors present within the bioreactor, and the signals generated are monitored by a central microprocessor. Therefore, environmental conditions,
For example, without limitation, changes in pH, temperature and dissolved gases can be automatically monitored and altered as needed. In addition, the state of cell growth is indirectly assessed by detection of turnover as a function of time (eg pH, _O2 , _CO2 , lactate and glucose consumption). In addition to control of processing conditions by a central microprocessor, the tissue engineering module itself may optionally include a secondary on-board microprocessor that operates in unison with the central microprocessor. The tissue engineering module microprocessor expands the data processing capabilities of the tissue engineering system by performing specific functions mounted directly on the tissue engineering module, thereby minimizing reliance on the central microprocessor. to

Various growth factors, cytokines, experimental drugs, drugs, chemicals, media and any combination thereof may be loaded and stored in any reservoir located on the tissue engineering module and pre-programmed. may be automatically transferred to one or more bioreactors according to a specific sequence or as required by the developing tissue implant. The individual tissue engineering modules are removable from the system for transport without compromising the sterility of the tissue engineering constructs present within the bioreactor. Such removal does not affect the processing of any other modules present within the tissue engineering system. Additionally, the tissue engineering module can be considered disposable after completion of the tissue engineering procedure. This is because doing so prevents contamination from previous uses.

In various embodiments of the invention, the device and system can be used to digest tissue obtained by surgical biopsy. In another embodiment, cells can be filtered and specific populations can be selected and isolated. In another embodiment, digested cells can be grown to expand the population of cells. In still further embodiments, cells can be seeded and cultured on a desired scaffold or substrate (also called matrix). In still further embodiments, cells may be differentiated onto and/or across a desired scaffold or substrate until appropriate tissue formation is obtained. In still further embodiments, tissue may be stimulated to promote tissue maturation.
In yet another embodiment, tissue implants are produced that are suitable for reconstructive surgery. In still further embodiments, cell sampling can be performed at each stage of cell growth and developmental progression in an aseptic manner without detrimental effects on the culture itself. If desired, each of the foregoing embodiments can be performed singly or in sequence. Tracking of such processing events can be performed by a central microprocessor and/or module-based microprocessors for incorporation into quality control records.

In one embodiment, the tissue engineering system is optionally disclosed in Applicant's U.S. Pat.
323,146, the contents of which are incorporated herein by reference, are used to enhance biological potency. Briefly, Skelite™ is an isolated, bioresorbable, biomaterial compound containing calcium, oxygen and phosphorus, with a fraction of at least one of the elements being about 0.0.
Substituted by elements with ionic radii between 1 and 0.6 Angstroms. In one embodiment, Skelite™ enhances cell growth through its use as a coating on the walls of a bioreactor, as a thin film on a growth substrate, or as a three-dimensional and thereby high surface area growth scaffold. can be used to The use of Skelite™ in the expansion phase can be demonstrated to do the following.
increasing the rate of proliferation,
increasing the cell yield after the expansion step;
reduce the surface area required for target cell yield,
Reduces the problematic propensity for cell phenotypic dedifferentiation during proliferation and enhances binding of growth factors to growth substrates.

In a further embodiment, Skelite™ can be used as a resorbable scaffold to enhance cell differentiation and subsequent formation of tissue constructs. The use of Skelite™ in the differentiation stage can be demonstrated to do the following.
Increase productivity by improving the reliability of the differentiation stage.
Improves tissue construct integrity and thus biological viability.
Allows flexibility in placement of constructs based on different scaffold formats.
It allows the stages of proliferation, differentiation and tissue formation to occur on a common substrate.
Enhances binding of growth factors to differentiation scaffolds.
Improves handling properties of tissue constructs during surgical implantation.

In another aspect, the invention provides for the digestion, expansion,
Methods and systems are provided for the preparation of tissue constructs through automated steps of seeding and differentiation, thereby eliminating immunological and disease transmission problems. Implants may be formed from controlled cultures of various cell types including, but not limited to, chondrocytes, stromal cells, osteoblasts, neuronal cells, epithelial stem cells and mixtures thereof.

The system of the invention in certain embodiments incorporates one or more removable, portable, and independently operable tissue engineering modules, which include one or more bioreactors, media reservoirs, and support the liquid/medium flow system. Each module, and thus bioreactor(s), is under automated control of a central microprocessor. This module and associated bioreactor can be configured for a variety of specialized applications, including but not limited to the following.
Sterile receipt/storage of tissue biopsies,
automatic mixing and delivery of digestion reagents,
automatic monitoring of the digestive process,
Digestion of biopsy tissue to obtain dissociated cells,
cell sorting and selection, including safe waste collection;
cell washing and cell collection,
cell seeding onto or into a growth substrate or scaffold;
automatic mixing and delivery of proliferation reagents;
proliferation of cells to expand the cell population;
automated monitoring of cell conditions, including detection of confluence;
controlled release of cells from a growth substrate or scaffold;
repeated amplification steps for selected surface area sizes to increase cell number;
channeling cell populations to one or more tissue engineering scaffolds or matrices;
seeding cells onto or into a tissue engineering scaffold or matrix;
automatic mixing and delivery of differentiation reagents;
automatic monitoring of cell/tissue culture conditions,
cell differentiation, which allows for the specialization of cell activity;
tissue formation,
mechanical and/or biochemical stimuli that promote tissue maturation;
Collection of tissue engineered constructs/implants for reconstructive surgery and storage and transport of cells and/or transplantable tissue.

When two or more bioreactors are provided within the system, either supported directly within the housing of the system or supported on tissue engineering modules insertable into the housing, this The bioreactors may be provided connected so as to be operable in series and individually and controlled by a microprocessor or depending on the programming of the user of this microprocessor and the desired results to be achieved. may be operated and controlled independently of each other. Additionally, if more than one bioreactor is provided within the system, the bioreactors and internal chambers may be connected such that there is an exchange of cells and/or tissue from bioreactor to bioreactor.

The bioreactor may be manufactured in different sizes and shapes as needed to support different numbers and sizes of growth and differentiation scaffolds or substrates. This bioreactor may be incorporated as part of the structural component of the tissue engineering module. Alternatively, the bioreactor may be removable as a separate component to the remaining components of the tissue engineering module. When present as separate components, the bioreactor may be separately packaged in sterile packaging and connected to the tissue engineering module using sterile access techniques at the point of use. In addition, this sterile access technique allows the bioreactor to be detached from the module upon completion of the tissue engineering process to facilitate transport to a working room in preparation for retrieval of newly formed implantable tissue constructs. it becomes possible to

The bioreactor and/or tissue engineering module may be rotated or agitated within the overall tissue engineering system via control actuators. Rotation may allow the advantageous use of gravity to perform specific biotreatment procedures such as sedimentation-based cell seeding and liquid exchange within the bioreactor.

The tissue engineering module may be bar coded or contain a memory chip for rapid and precise transport both within the tissue engineering system and externally as part of a clinical or experimental environment. may be provided. Transport technologies such as those incorporated into tissue engineered devices also enable electrical transport through clinical-based information systems for patient documentation. This allows the tissue engineering module, and therefore the associated cell or tissue implant, to be accurately coded to ensure correct patient dosing and to document this process for hospital billing purposes. The modules and/or bioreactors may also utilize barcodes and/or memory chips in a similar fashion for rapid and accurate patient and sample transport.

According to one aspect of the invention, an automated tissue engineering system comprising:
a housing;
at least one bioreactor supported by the housing, the bioreactor facilitating physiological cell function and/or production of one or more tissue constructs from a cell and/or tissue source;
a liquid containment system supported by the housing and in fluid communication with the bioreactor;
one or more sensors coupled with one or more of the housings, bioreactors or fluid containment systems to monitor parameters associated with the physiological cell function and/or production of tissue constructs;
a microprocessor coupled to one or more of the sensors.

According to another aspect of the invention, an automated tissue engineering system comprising:
a housing;
at least one tissue engineering module removably connected within the housing, the tissue engineering module comprising a support structure holding at least one bioreactor; a liquid in fluid communication with the bioreactor; Equipped with a containment system and one or more sensors for monitoring parameters associated with the cell culture and/or tissue engineering function, the bioreactor is a source of physiological cell function and/or cells and/or tissue. facilitating the production of one or more tissue constructs from
a tissue engineering module;
a microprocessor disposed in the housing and coupled to the tissue engineering module for controlling operation of the tissue engineering module;
Prepare.

According to a further aspect of the invention, a portable and sterilizable tissue engineering module comprising:
a structural support holding at least one bioreactor, the bioreactor facilitating cell culture and tissue engineering functions;
a liquid containment system in fluid communication with the bioreactor;
and one or more sensors for monitoring parameters associated with this cell culture and tissue engineering function.

In aspects of this embodiment, the bioreactor includes a bioreactor housing having one or more inlet ports and one or more outlet ports for the flow of media and cells and/or tissue to receive the cells. At least one chamber is provided within the bioreactor housing to facilitate culture and tissue engineering functions. The chamber may be selected from the group consisting of a cell culture and/or proliferation chamber, a cell differentiation and/or tissue formation chamber, a tissue digestion chamber, and combinations thereof. Additionally, the chamber houses one or more substrates and/or scaffolds. In embodiments of the invention, two or more chambers may be provided and may be operably connected within the bioreactor. or 2
One or more bioreactors may be operable independently or cooperatively. In still further aspects, the chambers and/or bioreactors are operatively connected to provide fluid, cell and/or tissue exchange between the chambers and/or bioreactors. Scaffolds for use in the present invention are selected from the group consisting of porous scaffolds, porous scaffolds with gradient porosity, porous reticulated scaffolds, fibrous scaffolds, membrane-reinforced scaffolds, and combinations thereof. Chambers may also be further subdivided into zones. For example, the differentiation and/or tissue formation chamber may be zoned to contain several scaffolds. Funnels or similar passages may be provided between chambers within the bioreactor. Additionally, one or more filters may be provided at any location within the bioreactor.

According to another aspect of the invention, storage of tissue biopsies, digestion of tissue biopsies, cell sorting, cell washing,
cell culture selected from the group consisting of cell enrichment, cell seeding, cell proliferation, cell differentiation, cell storage, cell transport, tissue formation, implant formation, transplantable tissue storage, transplantable tissue transport and combinations thereof; /or a bioreactor that provides an environment for tissue engineering functions.

According to yet another aspect of the invention, a bioreactor for facilitating and supporting cell function and the production of implantable tissue constructs, the bioreactor comprising:
a bioreactor housing;
one or more inlet ports and one or more outlet ports for the flow of media;
at least one chamber defined within the bioreactor housing for facilitating and supporting cell function and/or production of one or more tissue constructs from a cell and/or tissue source;
and one or more sensors for monitoring parameters associated with the cell function and/or the production of tissue constructs within the at least one chamber.

In embodiments of the invention, the bioreactor housing includes a lid, which may be a removable lid or a lid integral with the bioreactor housing.

The cells and tissues may be selected from bone, cartilage, related bone and cartilage precursor cells, and combinations thereof. More particularly, cells suitable for use in this bioreactor, module and system of the invention include, but are not limited to, stem cells derived from bone, bone marrow or blood, including embryonic stem cells, adult stem cells, osteoblasts. , pre-osteoblasts, chondrocytes, nucleus pulposus cells, pre-chondrocytes, skeletal progenitor cells, and combinations thereof. The cells or tissue may be autologous, allogeneic, or xenogeneic to the recipient of the implant formed by the cell culture and tissue engineering features of the present invention.

According to another aspect of the invention is a tissue implant produced within the bioreactor of the invention.

According to yet another aspect of the invention is a tissue implant produced by the tissue engineering system of the invention.

According to another aspect of the present invention, a tissue-engineered implantable construct for the repair of bone trauma, wherein the implant is combined with active bone cells and a tissue-engineered mineralized matrix It includes a porous scaffold of bone biomaterial.

According to another aspect of the invention, a tissue engineering implant comprising:
a cartilage zone containing tissue-engineered cartilage devoid of any mineral-based scaffolding;
a bone biomaterial zone comprising a porous scaffold; and a boundary zone between the cartilage zone and the bone biomaterial zone.

When implanted in vivo, the cartilage zone promotes lateral integration with the host cartilage, while the bone biomaterial zone promotes lateral and vertical integration with the subchondral bone plate. The boundary zone provides a structural connection between the cartilage zone and the bone biomaterial zone. This cartilage zone can further incorporate a secondary non-mineral scaffold that aids in the formation of tissue-engineered cartilage to match the specific anatomical features present at the site of implantation. Allows for the development of a formed surface profile.

According to another aspect of the invention, a method for digesting a tissue biopsy comprising:
filling a tissue biopsy into a bioreactor connected to a media reservoir and a flow system, the bioreactor communicating to a microprocessor one or more having a sensor of
providing tissue-digesting enzymes; and monitoring and maintaining appropriate digestion conditions within the bioreactor for a time sufficient for the desired level of tissue digestion.

According to another aspect of the invention, a method for proliferation of cells comprising:
Seeding cells onto a growth substrate or scaffold supported within a bioreactor connected to a media reservoir and flow system, the bioreactor communicating to a microprocessor the physiological conditions within the bioreactor. and monitoring and maintaining appropriate culture conditions within the bioreactor for a time sufficient for desired levels of cell growth.

According to another aspect of the invention, a method for differentiation of cells comprising:
Seeding cells onto a differentiation substrate or scaffold supported within a bioreactor connected to a media reservoir and flow system, the bioreactor communicating to a microprocessor the physiological conditions within the bioreactor. and monitoring and maintaining appropriate culture conditions within the bioreactor for a time sufficient for the desired level of cell differentiation.

According to another aspect of the invention, a tissue biopsy is digested to provide primary cells, including progenitor cells, such as stem cells, and then the cells are grown and differentiated to enable the formation of tissue implants. A method for causing the
filling a tissue biopsy into a bioreactor connected with a media reservoir and flow system, the bioreactor detecting and relaying physiological conditions within the bioreactor to a microprocessor; having one or more sensors of
providing tissue digestive enzymes;
monitoring and maintaining appropriate digestion conditions within the bioreactor for a time sufficient to obtain dissociated cells;
seeding the dissociated cells onto a growth substrate or scaffold supported within a bioreactor connected to a media reservoir and flow system, the bioreactor communicating to a microprocessor the physiology within the bioreactor; having one or more sensors for detecting a condition;
monitoring and maintaining appropriate culture conditions within the bioreactor for a time sufficient for desired levels of cell growth and amplification;
releasing the expanded cells from the growth substrate or scaffold;
seeding the expanded cells onto a differentiation substrate or scaffold supported within a bioreactor connected to a media reservoir and a flow system, the bioreactor communicating with a microprocessor to the having one or more sensors to detect and relay physiological conditions; and monitoring and maintaining appropriate culture conditions within the bioreactor for a time sufficient to obtain a tissue implant. do.

According to another aspect of the invention, a method for providing a skeletal implant, the method comprising:
Seeding osteogenic and/or osteoprogenitor cells into a porous scaffold of bone biomaterial supported within a bioreactor connected to a medium reservoir and flow system, the bioreactor comprising a micro having one or more sensors for detecting physiological conditions within the bioreactor to a processor, and the osteogenic activity throughout the scaffold to provide a tissue implant for orthopedic applications. This includes monitoring and maintaining appropriate conditions within the bioreactor for a period of time sufficient to allow the cells and/or osteoprogenitor cells to proliferate and/or differentiate.

According to yet another aspect of the invention, a method for providing a cartilage implant comprising:
This method is
seeding chondrogenic cells and/or chondroprogenitor cells onto a biomaterial porous scaffold supported within a bioreactor connected to a media reservoir and a flow system, the bioreactor being controlled by a microprocessor; having one or more sensors to detect physiological conditions within the bioreactor; and the chondrogenic cells and/or throughout the scaffold to provide a cartilage implant.
or monitoring and maintaining appropriate conditions within the bioreactor for a period of time sufficient to allow chondroprogenitor cells to proliferate and/or differentiate.

According to yet another aspect of the invention, a method for washing cells, the method comprising:
filling the chamber with a cell suspension containing one or more undesirable chemicals;
said cell suspension from said chamber through a cross-flow filtration module comprising a membrane impermeable to said cells but permeable to said undesired chemicals to provide a washed cell suspension; and collecting the washed cell suspension.

According to yet another aspect of the invention, a method for enrichment of cells comprising:
filling the chamber with a cell suspension having an excess cell suspension volume;
Continuously circulate the cell suspension from the chamber through a cross-flow filtration module that is impermeable to the cells but has a membrane that allows the excess cell suspension volume to be removed and collected. systematically recycling.

According to yet another aspect of the invention, a method for providing an implant for regenerating the inner nucleus of an intervertebral disc, comprising:
seeding nucleus pulposus cells within a porous scaffold of a biomaterial scaffold supported within a bioreactor connected to a media reservoir and flow system, the bioreactor communicating the bioreactor to a microprocessor; Having one or more sensors for detecting physiological conditions within the reactor and allowing proliferation and/or differentiation of the nucleus pulposus cells and expression of extracellular matrix components characteristic of the nucleus pulposus. monitoring and maintaining appropriate conditions within the bioreactor for a period of time sufficient to do so.

According to a still further aspect of the invention, a method for the preparation of quality assessment samples for use in clinical tissue engineering, the method comprising:
Parallel preparation of primary and secondary implants using the system of the invention described herein, wherein the primary implant is for implantation and one or more secondary implants are for the capacity of the primary implant. including preparations that are test purposes to infer the

In various embodiments, the tissue engineering system of the present invention is under the control of one or more microprocessors, which perform functions such as tissue digestion, cell proliferation, cell differentiation and/or tissue construct formation within the bioreactor. can be pre-programmed to allow the user to select a particular type of environment (or result of the environment). This eliminates operator intervention and reduces the potential for inadvertent contamination.

The tissue engineering system of the invention may be provided as a "kit." In this manner, the device, tissue engineering module(s), bioreactor(s) and its various components can be packaged and sold along with equipment and quality control techniques.

This system of the present invention is ideal for clinical use in hospitals and particularly in surgical situations where tissue engineering implants are desired due to trauma and/or disease. Using this system, tissue engineered implantable constructs can be safely prepared from autologous tissue, allogeneic or xenogeneic cells obtained from patient biopsies. The specifications of such tissue engineered implantable constructs can be adapted to the type, size and conditions of the implantation site. In addition, implants such as those produced by the system of the present invention contain active cells that facilitate integration into the host and thus promote patient recovery.

In fact, using the autologous cell model, tissue biopsies can be obtained from a patient and placed directly into a bioreactor residing in the tissue engineering module in the operating room. A particular bioreactor design is selected depending on the type and size of tissue construct desired. Upon completion of the tissue engineering process, the tissue construct produced, still contained in the sterile bioreactor, may be transported to the operating room for implantation back into the patient. This system is ideal for providing "customized" autologous tissue implants in a safe and therapeutically effective manner.

The systems and methods of the present invention are not limited to providing automated cell culture techniques. The tissue engineering system described far exceeds cell expansion used in cell therapy. Tissue engineering systems can be used to create functional tissue constructs in which the cells present are active, differentiated and already expressing extracellular matrix. As a result, tissue constructs so produced are in a highly developed state, thereby accelerating the rate and quality of tissue repair at the implantation site.

The system of the invention is also suitable for pharmacological studies. In particular, this system finds application in the area of drug development. New potential drugs and molecules can be tested on cells and tissues to determine their effects on cellular events and tissue development. Such tests can be performed on the patient's own cells/tissues to assess and possibly avoid adverse side effects prior to administration. Alternatively, specialized cell lines or tissues can be used in this system as important tools in the drug discovery process. This system is programmed to monitor and assess various physiological conditions of cells/tissues present within the bioreactor, thus providing a robust indication of the biological effect of a selected drug or molecule. can do.

The system can also be used for research and research and development in conditions that are difficult to implement using conventional tissue engineering techniques and/or require extensive diagnostic documentation. For example, microgravity studies involving tissue engineering are difficult to perform due to the unique properties of this environment. Traditional cell and tissue culture techniques cannot easily be performed in this environment due to fluid containment problems and the lack of gravity-based transport of cells. The systems and methods of the present invention are readily adaptable to microgravity environments. Because the system is completely sealed to prevent fluid loss and cell migration, part of the tissue engineering process can be accomplished with controlled fluid flow.

Other features and advantages of the invention will become apparent from the following detailed description, examples and drawings. However, it should be understood that this detailed description, specific examples and drawings show embodiments of the invention by way of illustration only.
Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

The invention will be further understood from the following description with reference to the drawings.

The general methodology for clinical tissue engineering applied to the example of cartilage repair using autologous chondrocytes is illustrated. 1 shows an integrated tissue engineering device of the present invention. 3 shows a further embodiment of the tissue engineering device of FIG. 2; 3 shows a further embodiment of the tissue engineering device of FIG. 2; Figure 3 shows a cross-sectional view of the tissue engineering device of Figure 2, illustrating some of the internal components and tissue engineering modules for insertion into the device; Figure 3 shows an enlarged cross-sectional view of the tissue engineering device of Figure 2 illustrating an inserted tissue engineering module; FIG. 12B shows an enlarged perspective view of the tissue engineering module and the interface with the components of the device housing. 7a shows an enlarged perspective view of the bioreactor and pump unit and 7b shows an enlarged perspective view of the pump unit and connected pump tubing. 8 shows a perspective view of the backside of the tissue engineering module of FIG. 7 and the internal configuration of the flow plate attached thereto. FIG. FIG. 12 shows an enlarged perspective view of the mixing and microfilling components connected to the bioreactor design with the device. Schematic representation of basic tissue engineering fluid flow. FIG. 11 shows a further embodiment of a schematic diagram of a basic tissue engineering fluid flow; FIG. Alternative bioreactor, growth substrates or scaffolds, differentiation scaffolds, and process monitoring designs applicable to different tissue engineering scenarios are shown. FIG. 13B shows an enlarged perspective view of the bioreactor of the tissue engineering module, illustrating the internal configuration of the bioreactor and the fluid flow passages. A further embodiment of the bioreactor of the tissue engineering module is shown, illustrating the internal configuration of this bioreactor. A rotatable bioreactor design is shown. Figure 10 shows a sterile sampling embodiment of the tissue engineering module; FIG. 11 shows a further embodiment of a tissue engineering fluid flow schematic. FIG. 11 shows a still further embodiment of a tissue engineering fluid flow schematic. FIG. Suitable bioreactor designs for tissue engineering and cell harvesting are shown. A suitable bioreactor design for cell growth is shown. A suitable bioreactor design for cell differentiation and tissue construct formation is shown. FIG. 11 shows a still further embodiment of a tissue engineering fluid flow schematic. FIG. Figure 10 shows a further embodiment of a tissue engineering module with separate bioreactors for tissue engineering/cell harvesting, cell growth and cell differentiation and/or tissue formation.

The present invention provides cell growth,
It relates to an integrated, automated tissue engineering device for ex vivo processing of cells, in particular autologous cells, that allows cell differentiation and tissue formation. Tissue constructs developed within this device may be incorporated into a host to aid in tissue remodeling procedures and subsequent patient recovery. Additionally, the present invention provides automated methods for tissue engineering using a variety of cells from a number of different sources (e.g., autologous cells obtained via patient biopsy, allogeneic cells or xenogeneic cells). cell). Additionally, the cells may be progenitor cells, primary cells, cells from immortal cell lines, and combinations thereof.

The general methodology and principles for clinical tissue engineering incorporating tissue engineering systems and methods of the present invention are illustrated in FIG. 1, using autologous cartilage tissue engineering as a representative example. . In such instances, cells (i.e., chondrocytes) are obtained from a surgical biopsy of a patient and seeded onto a suitable substrate or scaffold (i.e., Skelite™ support), either manually or automatically. do. The chondrocytes and scaffolds reside within the bioreactor portion of the automated tissue engineering module, with the module forming part of the clinical base station of the tissue engineering system. A central microprocessor resides within this tissue engineering system to control and customize the internal environment of the bioreactor, thereby facilitating tissue growth therein, within and within supports for generating implants. Stimulation of cell proliferation occurs on the body. The sensors in the bioreactor are
Feedback is provided to the microprocessor to ensure that the cells are seeded, expanded and differentiated in the desired controlled manner to provide an autologous tissue implant. Once the implant is produced, it is moved from the bioreactor for surgical implantation to the patient. The system of the present invention provides an advantageous method for providing autologous tissue engineering implants in a sterile, safe, convenient and effective manner. Furthermore, the ability to prepare tissue engineered implants in a clinical setting allows considerable freedom in where to perform the tissue engineering process. Although the system can be used in centralized locations, the design and operation of the system allows for clinical use in regional centers. Such broad availability prevents transport of biological material to and from centralized cell/tissue processing facilities, thereby avoiding transport, tracking and regulatory problems, while The cost-effectiveness and effectiveness of tissue engineering processes are improved.

According to an embodiment of the present invention, a tissue engineering system, such as that shown in FIG. The system 100 (which may alternatively be referred to as a device) includes a housing 102 having an insertion slot 104 for receiving a tissue engineering module. This insertion slot 104 has a movable door 106 and a locking mechanism 108 . A user interface 110, such as a touch screen, keypad, or a combination of both, is provided for control of system operation and for presentation of system status. A data storage system 112 is present and allows recording of information via various media known to those skilled in the art (ie, ZIP, CDROM, diskettes, flash cards). A computer/communications link 114 provides the ability to upload new software, modify control parameters, download data, and troubleshoot and test the device using an external computer. This link also allows the system to be connected to existing electronic information systems in the clinic. The system 100 is powered at power input 116 . FIG. 3 shows a further embodiment of system 100 having several bay doors 106 to accommodate several tissue engineering modules. FIG. 4 shows a further embodiment of system 100 having bay doors 106 oriented in a horizontal fashion to allow preferential orientation of the tissue engineering module with respect to the gravity vector.

FIG. 5 shows the internal configuration of the system 100 presented in FIGS. 2 and 3, where the bay door orientation is vertical for vertical insertion of the tissue engineering module. Tissue engineering module 118 is shown for insertion into insertion slot 104 of bay door 106 . This tissue engineering module 118 is connected to the system housing 1 via a guide rail system 120 .
Slide into 02. Upon insertion, this module 118 includes one or more pump units 122 (i.e. peristaltic, piston, diaphragm or rotary pumps), electrical connectors 124 (i.e. DIN, AMP, PCB, breadboard sockets), and It engages a valve actuator 126 (ie, servo motor, linear drive, linear actuator). Any suitable guide system that allows the module to be properly inserted into the system may be contemplated as understood by those skilled in the art.

As better shown in FIG. 6, where tissue engineering module 118 is inserted into housing 102, a series of valve actuators 126 interface with the valves on this module (shown in greater detail in FIGS. 7 and 7a). to provide flow control. Electrical connectors 124 connect to module 118 via electrical backplane 130 .
and a central microprocessor unit (CPU) 128.
The CPU 128 controls operating procedures, liquid and gas transport, process data management, system status monitoring, user interfaces and external data communication ports. The CPU 128 provides control through active and passive electrical components present on the backplane 130 and electrical links with each inserted tissue engineering module 118 .

temperature sensor 132 (i.e. thermocouple, RTD or thermistor), gas sensor 134 (i.e. _O2 and _CO2 ) and environmental control unit (ECU) 136
is controlled by CPU 128 to maintain the environment (ie temperature and gas atmosphere) within housing 102 using standard methods known to those skilled in the art. This environment includes storage of reagents at refrigerated temperature (i.e. 4°C), normal body temperature (i.e. 37°C) stimulation, and module It can be adjusted to meet the requirements of the tissue engineering process, including the availability of gas mixtures for transport in and out of 118 . Gas conditions are monitored by a gas sensor 134 located within housing 102 and this data is sent to CPU 128 via electrical backplane 130 . Gas input to the ECU may be through a gas supply inlet 140 provided within the housing 102 constructed to a standard fit. In other embodiments, the gas may be housed within the ECU. Gases for use within the device include:
Examples include, but are not limited to, oxygen, carbon dioxide, nitrogen and mixtures thereof. To adequately contain such gases within housing 102, bay door 106 is configured to provide an airtight seal when closed. The housing 102 is insulated with an insulator 142, such as styrofoam, airgel, fiberglass, etc., to allow efficient regulation of the internal temperature (ie, 4° C. to 37° C.).

Although the tissue engineering system of the present invention is generally shown to comprise a box-like shaped housing, as long as the housing can accommodate the components as described herein,
It will be appreciated by those skilled in the art that it may be made from a variety of shapes. For example, this includes, but is not limited to, open shapes that may not require a top and/or sides.

This tissue engineering module 118 is illustrated in detail in FIGS. The tissue engineering module 118 comprises a rigid structural spine 200 to which a bioreactor 202 is secured. This bioreactor 202 is used for tissue digestion, cell culture, cell proliferation, cell differentiation,
A bioreactor housing having a lid 204 can be provided and customized for the substrate(s) or scaffold(s) contained therein to allow for tissue implant formation and combinations thereof. The bioreactor lid may be removable from the bioreactor housing or integral with the bioreactor housing. The bioreactor 202 is separately removable from the structural spine 200 and may be disposable. To enable such desorption, the bioreactor 202 and its structural spine 20
0 may use liquid disconnect fittings with provisions for self-sealing the input and output lines to avoid liquid loss and prevent contamination of the bioreactor contents. This entire tissue engineering module can be considered disposable after completion of the tissue engineering procedure. This is because this practice prevents contamination caused by previous use. Alternatively, only selected components of module 118 may be considered disposable due to contact with liquids, leaving non-contaminating components available for reuse.

As shown in FIGS. 7, 7a and 7b, the fluid containment system 206 is secured onto the structural spine 200 of the tissue engineering module 118. FIG. The fluid containment system 206 includes a sterile series of flexible reservoirs 208 for supplying and retrieving various types of tissue and cell culture fluids and chemicals to and from the bioreactor 202 .
and flexible tubing 210 . The reservoirs 208 may be of various configurations and numbers as desired, and may contain various types of cell and tissue culture media, growth factors, pharmaceutical agents, and may also be used to cleanse the bioreactor 202. Media and/or wash samples may be included. Liquid is charged to or removed from the liquid containment system 206 via a series of liquid access ports 212 . Lines 210 are present to provide fluid communication between various reservoirs 208 and liquid control components, such as liquid flow control valves 214 . This liquid flow control valve 214 is the valve actuator 1
26 is opened and closed. Similarly, this pump unit 122 interfaces with the disposable pump components present on this module. The components of these pumps are pistons,
It may be a diaphragm, rotary element or peristaltic tubing 218 . The pump unit 122 and valve actuator 126 reside within the housing 102 . Alternatively, the actuator and pump unit may form part of a tissue engineering module, allowing these components to be disposable after patient use. Liquid is moved out of reservoir 208 by the programmed action of pump unit 122 on pump line 218 . The liquid enters the plastic liquid reservoir 2 via line 210.
08 to liquid valve 214 . A liquid flow plate 220 (shown in FIG. 8) directs liquid flow between different flow control valves 214 and pump lines 218 of the pump unit 122 . The liquid is returned to the selected empty reservoir 208 for storage. A flexible printed circuit board (PCB) 222 provides an electrical interface for electrical components (ie, sensors) present on structural spine 200 and/or bioreactor 202 . When the sensor indicates that the monitored parameter (eg, pH) is outside of acceptable levels, the CPU triggers a control intervention to replace the medium in the bioreactor.

The tissue engineering module may optionally include a microprocessor 224 that allows data to be processed and stored directly in the module. This information is communicated to the central CPU 128, while the module is inserted into the housing 102 and retained in electrical memory for later access once the module is removed. In addition to the data stored via the microprocessor or memory chip present in the tissue engineering module, this module also optionally stores bar codes 226, A magnetic strip 228, electrical memory (not shown) and/or ID tag 230 may be provided.

As shown in FIG. 8, fluid flow plate 220 is secured to structural spine 200 of tissue engineering module 118 . Techniques for detachment of the liquid flow plate may be, but are not limited to, press fit, snap fit, ultrasonic welding, solvent bonding, etc., and the techniques employed ensure that sealing of the assembly avoids liquid loss. It is understood that the Liquid flow plate 2, as shown in the exploded view of FIG. 8a
20 has an integral fluid path 232 for providing a means of directing flow associated with actuation of fluid valve 214 . New flow passages may be connected through modifications to existing passages in flow plate 220 . In one embodiment, this liquid plate 220 may be integrally formed into the structural spine 200 to form a single component. Heating and mixing of liquids to ensure that liquids directed to the bioreactor are at the correct temperature and are well mixed, thereby not disrupting ongoing biological processes in the bioreactor A chamber 234 is provided. Additionally, a thermoelectric element 236 is present on the tissue engineering module 118 to change the temperature within the bioreactor 202 relative to the module's operating temperature, as dictated by the operation of the ECU 136 . While such temperature changes may be necessary to stimulate apparent physiological conditions within the bioreactor, the remaining components of this tissue engineering module, particularly reagents and/or samples, are Low temperature (ie refrigerated) to maintain physical, chemical and/or biological viability. Powering and control of the thermoelectric elements is performed by the CPU 128 . In addition to sensors present in the bioreactor, sensors 238 present in the tissue engineering module provide feedback to CPU 128 . The sensor and thermoelectric connections are made via electrical cabling 240 and connector 124 .

FIG. 9 shows the mixing and microfilling aspects of tissue engineering module 118 . The bioreactor 202 has a mixing actuator 262 and a mixing drive 260 operably connected to a mixing diaphragm 264 . This mixing diaphragm is incorporated as part of bioreactor 202 or bioreactor lid 204 as shown. In operation, this mixing drive 260 , in combination with mixing actuator 262 , provides translation or pulsation of the mixing diaphragm that results in controlled mixing of the contents of bioreactor 202 . Ideally, the nature of the mixing is such as to avoid high liquid shear that would compromise the physical integrity of the cells present within the bioreactor. For certain tissue engineering protocols, moderate levels of liquid shear are actually advantageous for successful development of tissue constructs. In addition to the mixing components, there are impact drives 266 and impact actuators 268 . These components serve to apply a controlled impact to the bioreactor assembly at the end of the growth procedure to help release the cells from the growth substrate or scaffold present within the bioreactor. Also provided is a microfill drive 270 operably connected to the microfill actuator 272 and the microfill diaphragm 274 . This micro-filled diaphragm 274 is incorporated as part of the bioreactor 202 or bioreactor lid 204 as shown. The position and orientation of the micro-filled diaphragm is determined by the bioreactor 202
such that it allows intimate contact with the substrate or scaffold present in the cell and any associated cells or tissues. The application of microloading is known to be advantageous for certain tissue engineering protocols. Mixing drive 260, impact drive 266 and microfill drive 270 can be any set of electromechanical devices, e.g., solenoids,
It may be a linear drive, a rotary drive or a piezoelectric electric component. Additionally, the mixing drive 260 , impact drive 266 , microfill drive 270 and associated actuators can be mounted on the housing 200 . Alternatively, the drive and actuator may be mounted on the tissue engineering module or bioreactor, provided the design of the drive is consistent with the disposable nature of the tissue engineering module. In addition to providing mechanical stimulation, the bioreactor may also be configured to induce electrical and/or chemical stimulation of tissue constructs. In particular, an electric field may be generated in the region of this bioreactor to enhance cell trafficking and/or tissue formation. Methods of generating electric fields are known to those skilled in the art and include, but are not limited to, supplying electric coils.

Figure 10 illustrates a schematic of the basic fluid flow for the tissue engineering module;
There is a single cell or tissue culture chamber present in bioreactor 202 (see descriptions of FIGS. 12 and 17 for more information on multi-chamber bioreactors). The flow path is between bioreactor 202 and reservoir 208 .
, which supplies liquid and collects waste. This liquid access port 21
2 can be used to load reagents or withdraw samples or discard liquids. Flow is caused by operation of pump unit 122 in the direction of flow defined by operation of a particular diversion control valve 214 . Perfusion into the bioreactor can be either continuous or pulsatile, provided that the flow involved does not create high liquid shear in the region containing the cells. This is because such conditions can damage cells or nascent tissue constructs. recirculation loop 280
is provided, the liquid content of the bioreactor can be monitored or altered by an external component, such as an in-line gas exchange membrane 282, without requiring delivery of fresh liquid from the liquid reservoir 208. be possible. Components of the tissue engineering module 118 that correspond to storing liquids (i.e., reservoirs 208) are refrigerated at approximately 4° C. and are otherwise exposed to elevated temperatures used to simulate body temperature (i.e., , 37° C.) to facilitate storage of liquids. According to a preprogrammed routine, CPU 128 controls the operation of liquid valve(s) 214 to pump liquid stored in reservoir 208 through pump unit 122 prior to entry into bioreactor chamber 300 . heating and mixing chamber 234 (shown in detail in FIGS. 13, 14 and 15). Liquid is supplied to the bioreactor via inlet port 302 and removed via outlet port 304 . To simulate normal body temperature for optimal cell and tissue culture performance, bioreactor 202, pump unit 122 and heating and mixing chamber 234 are integrated with thermoelectric element 2
It is maintained at about 37°C by the operation of 36. Those skilled in the art will appreciate that other thermal conditioning devices can be used to obtain the desired thermal profile for tissue engineering module 118 .

FIG. 11 illustrates a variation of the basic liquid flow schematic, in which the liquid flow control valve is replaced by a multi-pump unit 122. FIG. This configuration provides increased operational redundancy and reduced component count. Operation of such a system requires that any dormant pumping units block unregulated through flow. This is because such phenomena impair the controlled delivery of liquids.

Figures 12a-12d illustrate various bioreactor configurations and alternate modalities for substrates and scaffolds used in the proliferation and differentiation processes involved in engineering tissue engineering systems.
Figure 12a shows a series of interchangeable bioreactor designs addressing different bioprocessing scenarios. The Type I scenario shows a basic single chamber 300 within the bioreactor 202, which is fitted with a growth scaffold or substrate 310, or a differentiation scaffold or substrate (not shown), ideally Suitable for either proliferation or differentiation.
Cells are either manually seeded onto the scaffold 310 or automatically delivered through the fluid passageways of the tissue engineering module.

The Type II scenario involves a multi-chamber bioreactor that provides for the use of a scaffold 310 (or substrate) for cell population expansion and an implantable differentiation scaffold 312 to facilitate the formation of tissue constructs. This culture and/or growth chamber 300 is connected to a differentiation and/or tissue formation chamber 306 via a funnel 314 . This funnel functions to carry cells liberated from growth scaffold 310 to implantable differentiation scaffold 312 . The use of filters 316 at several locations within this bioreactor functions to regulate the size of cells or cell aggregates that can freely pass from one chamber to the next. A filter 316a is upstream of this growth scaffold for the purpose of regulating the entry of the cell population for the cell expansion process. Another filter 316b is again present upstream of the differentiation scaffold to control the cell population entering this step of the tissue engineering procedure. Additionally, an additional filter 31 covering the outlet port 304 to prevent loss of cells from the differentiation and/or tissue formation chambers during operations involving fluid transfer through the bioreactor.
6c exists. This filter 316 may be a filter membrane or mesh or similar type filtering material, as is known to those skilled in the art.

The type III scenario combines tissue digestion with subsequent proliferation, differentiation and tissue architecture formation. In this scenario, a tissue biopsy 320 is loaded into a digestion chamber 322 present within bioreactor 202 . Digestion of the tissue biopsy occurs through delivery of digestive enzymes to the bioreactor from one of the liquid reservoirs 208 present in the tissue engineering module. Dissociated cells exit the digestion chamber 322 under the influence of gravity sedimentation and/or fluid flows through the culture and/or growth chamber 300 and continues to collect on the growth scaffold 310 . Migration of tissue aggregates outside of digestion chamber 322 is impeded by the presence of filter membrane or mesh 316a in the flow path between digestion chamber 322 and culture and/or growth chamber 300 . After growth, the cells are released and placed in the cell funnel 3.
Transfer via 14 to the implantable differentiation scaffold 312 . Again, membranes or mesh filters are present both upstream 316b and downstream 316c of the implantable differentiation scaffold 312 to ensure that the correct cell population is seeded onto the scaffold and that cells are inadvertently lost to fluid migration. Ensure that it is not discarded during work.

In the aforementioned scenario, various configurations of growth substrates 310 or scaffolds are possible, for example, as illustrated in FIG. 12b. For example, one configuration is a porous scaffold 310a with a relatively even pore gradient. Pore gradient scaffold 310b is a porous scaffold with a pore gradient where the pore size decreases as cells migrate through the scaffold. by this,
At the end of the cell seeding process, a more even distribution of cells throughout the scaffold is promoted.
A reverse pore gradient scaffold 310c may also be used. Alternatively, fiber filter scaffold 3
10d may also be used, which is a typical fibrous matrix for organic compounds such as collagen. As a growth substrate, it is also possible to utilize a suspension containing microcarriers (eg, Cytodex™). Furthermore, this bioreactor
Optical probes 324 supported by CPU 128 (porous scaffold 310a and shown in combination).

As with growth, there are a variety of implantable differentiation scaffolds 312 that can be formed in a variety of configurations and from a variety of materials (i.e., inorganic mineral-based scaffolds such as calcium phosphate, organic biopolymers such as collagen). scaffolds, etc.) and used in tissue engineering processes. Figure 12c illustrates a multi-zone differentiation and/or tissue formation chamber 306, which comprises up to three implantable differentiation scaffolds 312, all of which can be processed simultaneously toward tissue construct formation. This allows the preparation of implantable tissues of various sizes and the use of alternative implantable differentiation scaffolds to assess and maximize tissue yield. For example, scaffold 312a may be a porous network formed from a bone biomaterial such as Skelite™ for use in bone and cartilage applications where the tissue construct requires firm tissue anchorage within the bone. is. This scaffolding 312a can be further strengthened by the use of a scaffolding membrane or mesh 326 that surrounds the implant to form a membrane-enclosed scaffolding 312b, resulting in a scaffolding during the cell seeding process. Cell loss from 312a is minimized, thereby making the tissue engineering process more efficient. The membrane may preferably enclose the scaffold only partially, or may enclose the scaffold more completely. Although the primary purpose of this scaffolding membrane or mesh 326 is to contain cells on and within the implantable differentiation scaffold 312, careful selection of the properties of this scaffolding membrane or mesh 326 will allow one skilled in the art to As will be appreciated, the passage of certain molecular entities is allowed or restricted which can have a pronounced effect on the tissue engineering process at the cellular level.

A further embodiment is a gradient porosity and membrane enhanced scaffold 312c, which combines the advantages of the scaffold membrane or mesh 326 with a pore gradient. The gradient is configured to force cells to intentionally cluster on the top surface with minimal proliferation into the scaffold. The degree of porosity in this surface is believed to be beneficial for tissue stability and for supplying nutrients to the developing tissue through the scaffold surface. This approach results in the development of a bipolar tissue architecture with distinct stratified zones. This upper zone consists essentially of new tissue. The bottom zone contains essentially no cells or tissue and exists as an open porous scaffold. This intermediate boundary zone represents a structurally stable transition between the open scaffold and the new tissue layer. Such a bipolar tissue construct is ideal for the repair of focal defects in articular cartilage. Because this top layer
Although tissue engineered cartilage provides lateral integration with the host cartilage, this bottom layer provides lateral and axial integration with the subchondral bone. The integration of the bottom layer with the surrounding subchondral bone can be further enhanced by application of bone marrow to the open scaffold at the time of surgical implantation.
In cartilage repair applications, it is important that the mineral-based scaffold does not extend over the surface of the joint.
This is because mineral-based scaffolds can impair joint function. Therefore, a secondary non-mineral scaffold (not shown in the figure) was used in the superior zone of the new cartilage to a size sufficient to treat large cartilage lesions (i.e., up to 10 cm ² in diameter, and 2-3m thick
m) may assist in the formation of tissue constructs. Additionally, the secondary scaffold may be configured to produce a shaped construct with an articular surface profile that more closely matches the specific anatomy present at the site of implantation. Candidate materials for secondary scaffolds include synthetic biopolymers (e.g. PGA, PLA) or natural biopolymers (e.g.
alginic acid, agarose, fibrin, collagen, hyaluronic acid). These secondary scaffolds may be in the form of hydrogels or three-dimensional preformed scaffolds.

Alternative techniques for the preparation of bipolar tissue constructs are possible within this tissue engineering system.
The implantable differentiation scaffold 312 may be partially infiltrated with a bioresorbable polymer that limits cell seeding to specific areas of the scaffold. This creates a preferential zone of new tissue formation during preparation of the tissue construct. Upon implantation, the polymer is resorbed, thereby leaving voids in the porous scaffold that promote anchorage within the host tissue. A further configuration is differentiation and/or
or including an implantable scaffold with relatively open porosity located away from the presence of tissue formation chambers. During cell seeding, this open space provides a collection of cells that migrate through the open scaffold. As cells accumulate within the differentiation and/or tissue formation chamber, both the open spaces and portions of the scaffold become infiltrated by cells, thereby creating preferential zones for new tissue formation. The resulting tissue construct includes a nascent tissue zone, which includes neither a scaffold, an intermediate transition zone, a border zone containing both nascent tissue and scaffold, and a porous tissue that is open and essentially free of cells and tissue. It also lacks areas of sex scaffolding.

Figure 12d illustrates a bioreactor monitoring scheme in which sensors 216 (i.e. temperature, pH, dissolved gases, etc.)
128, lid 20 of bioreactor 202
incorporated into 4. Additionally, a CCD camera 330 may be used to monitor optical properties of the growth scaffold 310 (or substrate) for evidence of impending confluence (e.g., optical density and/or light scattering as a function of cell density). Often this allows the timing of cell release to be determined to maximize cell yield from the expansion process.

FIG. 13 better illustrates the flow paths and liquid circulation within bioreactor 202 . Bioreactor 202 is shown having an internal cavity defining inlet port 302 , outlet port 304 and basic chamber 300 . Liquid flows from liquid flow plate 220 through inlet port 302 into bioreactor 202 and out outlet port 304 . This bioreactor lid 204 is attached to the bioreactor 202 . A variety of different mounting hardware can be used to hold bioreactor lid 204 and bioreactor 202 together. This chamber 30
0 can be designed to fit one or more substrates or scaffolds 310 . Further, the bioreactor 202 may be subdivided into separate chambers that allow the processes of tissue digestion, proliferation, differentiation and tissue formation. each chamber spans the tissue engineering procedure,
It may consist of inlet and outlet ports that are independently controlled via flow control valves for greater control. Liquid circulation is accomplished through the actuation of one or more flow control valves 214 and pump units 122 based on control signals from CPU 128 . Depending on the particular valve that is actuated, operation of pump unit 122 moves liquid from one of liquid reservoirs 208 to bioreactor 202 or allows recirculation of liquid within the bioreactor. Recirculation is advantageous for biological processes that require stable dissolved gas concentrations. This is because recirculation allows the liquid to traverse the membrane facilitating gas exchange. The nature of this exchange is
Based on dissolved concentration in bioreactor to external conditions established by 36. The bioreactor lid 204 includes a sampling port 332 and sensor probe 2
16, which is operatively connected to the internal chamber of the bioreactor. Alternatively, the sampling port may be located elsewhere on the bioreactor housing. The sampling port allows removal or addition of substances to and from the bioreactor.
This sampling port may be replaced or reinforced with a gas exchange membrane if desired.

FIG. 14 illustrates a multi-chamber bioreactor 202 with the bioreactor lid 204 removed for clarity. This inlet port 302 is
Connected to tissue digestion chamber 322 . This tissue digestion chamber 322 configuration allows patient biopsies to be loaded into the bioreactor for subsequent automatic digestion to obtain dissociated cells. A circulation passageway within the digestion chamber facilitates gentle agitation of the biopsy and prevents a stagnant chamber that could potentially lead to excessive exposure of the biopsy tissue to circulating digestive enzymes. In addition, the digestion chamber inlet and/or outlet can accommodate various porous filter membranes or meshes 316 (not shown) to provide cell sorting and partially digested cells. It is possible to prevent the release of tissue aggregates. The bioreactor comprises a second chamber 300 containing a growth substrate or scaffold 310 to receive cells for growth. The growth scaffold may be formed in a variety of shapes that support both 2D and 3D growth,
and may consist of various biocompatible materials that promote cell proliferation, such as calcium phosphate biomaterials (eg Skelite™), biopolymers or natural matrices (eg collagen). Cells originating from the tissue digestion chamber 322 or optionally via the cell inoculation port 334 are dispersed on or within the growth substrate or scaffold 310 and proliferate, thereby resulting in subsequent cell differentiation and tissue formation. to expand the cell population for Note that this process may end after expansion if the goal is only to expand the population of cells without further differentiation. Implantable differentiation scaffold 312 is located at the base of bioreactor 202 in differentiation and/or tissue formation chamber 30 .
6. Like the proliferation scaffold 310, the implantable differentiation scaffold 312 may be formed in a variety of shapes and made of a variety of biocompatible materials appropriately selected to suit the biological requirements of the implantation site. For example, Skelite™ is an ideal candidate implant for skeletal sites).

During operation, cells are released from the growth substrate or scaffold 310 by automated procedures, such as delivery of enzymes (eg, trypsin) and timed application of impact to the bioreactor via impact drive 266 (not shown). be done. The cell suspension passes through the cell funnel 314 to the implantable scaffold 312 under the controlled flow conditions present in the bioreactor.
, on which the cells settle and initiate the differentiation and tissue formation procedure. At the end of the procedure, the tissue thus formed can be removed from the bioreactor for subsequent implantation. Those skilled in the art will appreciate that the specific embodiment of the bioreactor of Figure 14 is only an example of a representative design. The bioreactor can generally be configured in a variety of ways in terms of overall shape, size and internal configuration without detrimental effects on function. For example, gas exchange membranes 336 present in the bioreactor may be attached to one or more liquid delivery tubes 210 or flow plates 220 of tissue engineering module 118 .
may be separate and distinct components connected in-line with the . Additionally, the bioreactor chambers may be separated from each other via control valves to avoid the need for all liquids to pass through all chambers. If passageways are required between chambers, they may be opened to allow movement of liquids and cell suspensions. Examples of such variations that allow for increased flexibility in bioprocessing conditions and procedures are illustrated in FIG. Differentiation Scaffold 3 Implantable to Bioreactor Contents
Another configuration that would allow controlled exposure of 12 is the use of shuttles 318 to separate the implantable scaffolds until cell seeding is required as part of the differentiation process. This shuttle 318 moves the implantable scaffold from a protected position within the bioreactor into the fluid flow to allow cell seeding. Various configurations of this shuttle are possible, including:
Movement based on rotation, or the use of removable barriers separating the implantable scaffolds until cell seeding is required.

Figure 15 illustrates a rotating bioreactor design that utilizes the orientation of the gravity vector to achieve cell transport by sedimentation at different stages in the tissue engineering process. Note that although this figure illustrates rotation of the bioreactor, this technical goal can equally be achieved by rotation of the tissue engineering module, or indeed by rotation of the entire housing 102 . As shown in FIG. 15, this bioreactor 202
are tethered to a rotatable shaft 350 that is fixed to the structural spine 200 of the tissue engineering module 118 . This means that within the culture and/or growth chamber 300,
Bioreactor 202 so that cell seeding by sedimentation can occur on the selected growth surface.
provided with a rotation mechanism. The growth surface of this bioreactor consists of growth-enhancing biomaterials (
For example, it may optionally be coated with Skelite™, or a proprietary growth substrate or scaffold may be inserted into this chamber 300 to serve this role. As an alternative to using a digestion chamber 322, a second inoculation port 352 is provided on the side of bioreactor 202 to allow direct cell seeding. cells are
A relatively small growth surface 354 may be seeded first (Fig. 15a). Based on the passage of growth time or detection of confluence, cells may be automatically released and the bioreactor rotated by rotating shaft 350 so that cells released from this growth surface 354 are released from the surface. It deposits on an increased surface area of 356 (Fig. 15b), which allows for further proliferation. At the end of this secondary expansion step, the expanded cells are released and the bioreactor is spun again to allow seeding of the implantable scaffold 312 (Fig. 15c).
). Thus, this rotatable shaft 350 and associated flexible tube 358 allows the bioreactor 202 to rotate when necessary to maximize the use of gravity sedimentation in successive growth stages. become. If such conditions are desired, the use of a rotating shaft in a manner that agitates or permeates the bioreactor
It is within the scope of the present invention.

Referring now to FIG. 16, tissue engineering module 118 may be adapted to include techniques for aseptic sampling of suspended cells, tissue culture media, and/or waste products. In one embodiment, a syringe manifold 400 and sterile offloading port 402 are incorporated into the structural spine 200 of tissue engineering module 118 . Microporous tubing 406 connects the syringe manifold to bioreactor 202 via sampling port 332 . Syringe 404 is off-loading port 402 at manifold 400 to allow collection and removal of liquid samples or cell suspensions for subsequent analysis without compromising the manipulation, integrity or sterility of the tissue engineering process. connected to Another sampling technique is also provided in which a fusible bioreactor sampling line 408 is connected to the bioreactor lid 204 .
Because this line is physically connected to the interior of the bioreactor and is in close proximity to the biological event ongoing therein, this line is substantially identical to the components present within this bioreactor. Contains liquids. As a result, a representative sample of bioreactor fluid can be obtained by melting the end of a sampling line and then removing this line from the tissue engineering module for subsequent analysis. It will be apparent to those skilled in the art that such fusible lines can be used as a basis for sampling techniques through automated manipulation of hermetic components within housing 102 .

FIG. 17 illustrates a more complex fluid flow schematic for the tissue engineering module 118, where different requirements for digestion, proliferation and differentiation are provided by separate bioreactor chambers. These chambers may reside within a series of separate bioreactors or may be combined within a single bioreactor maintaining separate control over the conditions in each chamber. There is a tissue digestion chamber 322 containing a tissue biopsy 320 . There is a growth chamber 300 configured to receive cells from the digestion chamber 322 and to allow seeding of the growth scaffold or substrate 310 . an implantable scaffold 3 configured to receive an amplified cell population from the amplification chamber 300;
There is also a differentiation and/or tissue formation chamber 306 that allows 12 seedings.

Tissue engineering reagents (i.e. media, enzyme solutions, wash solutions, etc.) are stored in liquid reservoirs 208a.
Fill ~208e. Waste product is collected in liquid reservoir 208f, which is manually aspirated using access port 212f for the sampling process. Additional liquid reservoirs can be added to the liquid reservoir system 2 if required for different tissue engineering processes.
06 and may be connected to a tissue engineering module. Liquid flow through the system is controlled by liquid pumps 122a-122k, flow control valves 214a-2.
14c, and the operation of one-way flow valves 410a-410c (ie, liquid flow check valves). In addition, pumps 212a-212k may be active pumps or passive valves (open/close) according to control inputs from the central microprocessor.
closed). Filters 316a-316d are used to selectively control movement of the cell suspension within the system and to restrict passage of cell aggregates during the washing and migration stages of the tissue engineering process. . Dissolved gas levels in this medium are maintained via in-line gas exchange membranes 282a and 282b. Optional syringes 404a and 404b are present to allow cell collection or media sampling via sterile offloading ports 402a and 402b.

In operation, a tissue bipsy 320 passes through filters 316a and 316a.
inserted into tissue digestion chamber 322 between 16b. Digestion medium containing enzymes is pumped from liquid reservoir system 206 into tissue digestion chamber 322 to initiate the digestion process. Digestion can be enhanced by gentle agitation of the digestion medium within the digestion chamber via a mixing diaphragm to maximize reagent exposure to the biopsy. This digestion medium is
It can be continuously or periodically recirculated via pump 122g. During recirculation, this liquid flow is directed against the gravity vector to the bottom of the digestion chamber to suspend and invert the tissue biopsy, thereby maximizing the effectiveness of the digestion process. Filter 316a
prevents migration of cells and cell aggregates into the fluid passage. The recirculation passage includes an in-line gas exchange membrane 282a, which provides a constant level of dissolved gases in the digestion medium.
Introduction of the wash solution contained in the liquid reservoir system 206 to the bottom of the digestion chamber 322 flushes the digestion chamber to effectively wash the digestion medium from both dissociated cells and any remaining cell aggregates. do. After single or multiple wash procedures, application of the reservoir moves the cell suspension to either growth chamber 300 or syringe 404a as appropriate for external examination or analysis. Movement of partially digested tissue out of the digestion chamber is impeded by filter 316b, which is sized to allow passage of dissociated cells and retention of cell aggregates.

Cells obtained from a biopsy digestion process or available through direct filling of a cell suspension are forced onto the growth substrate or scaffold 310 present within the growth chamber 300 by liquid flow and/or gravity. Seeded by sedimentation. After a resting period to allow cell attachment to the growth substrate or scaffold 310 (e.g., attachment-dependent cells), the growth medium is
It is introduced into growth chamber 300 from liquid reservoir system 206 . This medium is periodically replaced with fresh growth medium from reservoir system 206 at specific times during the growth phase. During the medium change step, the liquid in this growth chamber is pumped by pumps 122g, 122h and 122.
i in addition to being continuously or periodically recirculated under the control of control valves 214a and 214b. The liquid delivery and recirculation passageway contains an in-line gas exchange membrane 282a, which provides a constant level of dissolved gases in the growth medium. During the medium replacement step, the supply of fresh medium from liquid reservoir system 206 is balanced by the removal of liquid to waste reservoir 208f via pump 122f. Thus, through a combination of periodic medium replacement steps and controlled recirculation, this tissue engineering system maintains optimal conditions within the growth chamber throughout the growth process.

Once the cell culture reaches confluence, the medium in growth chamber 300 is expelled by pump 122f into waste reservoir 208f. In this process, the removal of liquid from the growth chamber is by the incoming sterile air delivered through a sterile filter port (not shown) on the growth chamber or by PBS from the liquid reservoir system 206.
Equilibrated by the incoming wash solution. The cells are washed extensively by two successive washing steps with PBS wash solution to remove residual growth medium. The cells are subsequently liberated from the growth substrate or scaffold 310 by automated procedures such as delivery of enzymes (e.g., trypsin) and application of timed impacts to the bioreactor via an impact drive. . After cell release, enzymatic processing can be stopped by delivery of serum-containing medium, which inhibits enzymatic activity. Cell suspension is transferred from growth chamber 300 to filter 316 c for collection of cells for eventual seeding onto implantable scaffold 312 within differentiation and/or tissue formation chamber 306 . Filter 316c prevents the passage of cells, but medium is forced through valve 214b under the control of pump 122f to waste reservoir 208f.
allow you to proceed to The collected cells are then released from filter 316c by applying a reflux and delivered to either differentiation and/or tissue formation chamber 306 or optionally syringe 404b for external examination or analysis. do.

Cell seeding onto the implantable differentiation scaffold 312 is controlled by the filter 31 via pump 122j.
Accomplished by transferring cells from 6c to the top surface of this scaffold. Loss of cells away from the scaffold is minimized by the optional use of scaffold membrane or mesh 326 . After cell seeding, fresh differentiation medium can be introduced into differentiation and/or tissue formation chamber 306 through a secondary input by operation of pump 122k. This secondary input is located away from the area of the implantable scaffold that is seeded with cells, thereby minimizing the possibility of pure damage stress that could impair the formation of cell aggregates. This differentiation medium is periodically replaced with fresh differentiation medium from reservoir system 206 at specific time points during the differentiation phase. During the medium replacement step, the fluid in the differentiation and/or tissue formation chamber is continuously or periodically recirculated under the control of pump 122j or 122k as well as control valve 214b. The passageway for delivery of both fresh and recycled medium is equipped with an in-line gas exchange membrane 282b, which provides a constant level of dissolved gases in the differentiation medium.
During the medium replacement step, the supply of fresh medium from liquid reservoir system 206 is
Equilibration is achieved by removal of liquid via 2f to waste reservoir 208f. differentiation and/
Alternatively, environmental conditions within the tissue formation chamber are monitored and controlled for a period of time necessary for successful formation of the tissue construct, at which point the bioreactor differentiation and/or tissue formation chamber is opened for subsequent clinical use. This construct is recovered for use or research use.

FIG. 18 illustrates a variation to the fluid flow schematic of FIG. 17, in which the growth scaffold or substrate 310 in growth chamber 300 is replaced with a relatively large surface area planar growth substrate. The orientation of this substrate is such that cell sedimentation under gravity evenly distributes the cells over the growth surface. Provided the correct orientation of the growth chamber is maintained, the growth substrate may be in the form of a rigid polymeric culture plate or a flexible-walled container.

Figure 19 shows a tissue digestion bioreactor 500, which comprises:
An appropriately sized tissue digestion chamber 322 is provided to accommodate one or more tissue samples, such as a tissue biopsy 320 . This bioreactor 500 consists of four main components. A bioreactor base 502 that substantially forms a tissue digestion chamber 322, a moveable bioreactor lid 504, a port filter 316b, and an optional port filter 316a (not shown).

The bioreactor lid 504 includes a media port 506 and an air outlet port 508 with optional port filters 316a (not shown). Bioreactor base 500 houses filter 316b, which allows passage of dissociated cells out of tissue digestion chamber 322 and retention of tissue aggregates and biopsy debris via media port 510. .

After insertion of tissue biopsy 320, the bioreactor is either port 506 or port 510.
is filled under automatic control using an enzyme solution through the Addition of enzyme solution to tissue digestion chamber 322 is balanced by the escape of air through port 508 . Biopsy digestion is performed under continuous or intermittent recycling of the enzymatic solution, which keeps released cells in suspension and maximizes exposure of the biopsy to enzymatic reagents. During recirculation, this enzyme solution enters the bioreactor through port 510 and exits through port 506 . This creates a fluid flow path in the opposite direction to the gravity vector so that the biopsy is suspended and flipped to maximize the effectiveness of the enzymatic reagent. Digestion can be enhanced by gentle agitation of the digestion medium within the digestion chamber via a mixing diaphragm (not shown). Port 508 may be closed during any recirculation process. This is because air bubbles present in the liquid flow system are trapped in the top half of the bioreactor above inlet 512 of port 506 . Upon completion of the digestion procedure, application of a backflow of either air or media through port 506 moves the dissociated cells through port 510 to either a growth chamber or a cell collection container.

FIG. 20 shows growth bioreactor 520 that provides growth chamber 300 . The bottom of the growth chamber consists of a growth substrate 310 suitable for cell attachment and growth. To regulate or maintain the level of dissolved gases in the medium, a gas permeable membrane (not shown) may be incorporated into the top surface of the growth chamber, allowing the transfer of gases such as oxygen and _CO2 . Separation wall 522 divides the interior space of the growth chamber into a system of channels to provide a predefined passageway for media from inlet port 524 to outlet port 526 .
force to flow to

The design of the growth bioreactor design has several important operational features. A relatively uniform cell seeding can be obtained by injecting the cell suspension through the channel system. In addition, this channel configuration ensures that the flow of medium is well distributed over the growth surface, which can potentially compromise local cell vitality due to reduced nutrient supply or waste removal. areas of low flow are reduced. At the end of the growth procedure, continuous or intermittent recirculation of the appropriate enzymatic solution through this channel system results in a homogenous cell growth due to the effects of enzymatic reactions and the low levels of sheer stress caused by liquid flow. results in the desorption of Cell recovery is thus accomplished without the need for mechanical shaking or rotation of the growth chamber.

FIG. 21 shows a differentiation bioreactor 530 designed to promote cell differentiation and subsequent tissue construct formation. This bioreactor consists of four main components.
Bioreactor base 5 substantially forming differentiation and/or tissue formation chamber 306
32, removable bioreactor lid 534, permeable membrane tube 326,
and differentiation scaffold 312; The permeable membrane tube 326 tightly surrounds the mesh scaffold to form a tissue growth compartment 536 over the scaffold. The tissue growth compartment may extend within the scaffold depending on the pore size of the scaffold and the position of the scaffold within the membrane tube. The membrane tube is also secured to inlet 540 of port 542 so that the membrane is physically centered within differentiation and/or tissue formation chamber 306 . This divides the bioreactor into two independent compartments, a cell and tissue growth compartment 536 and an external cell-free media compartment 538, all of which are contained within the differentiation and/or tissue formation chamber 306 as a whole. be. The pore size of the membrane tube is selected based on being impermeable to cells but permeable to nutrients, wastes, growth factors, etc. within the culture medium. If desired, the pore size of the membrane can be selected in such a way as to exclude molecules of a certain molecular weight from passing through the membrane.

This bioreactor lid 534 has two air outlet ports 542 and 5
44, and one media inlet port 546. This bioreactor base 532 houses two additional ports 548 and 550 . This inlet port 546 is necessary for filling the tissue growth compartment 536 with a cell suspension and for perfusing the nascent tissue construct with culture medium. During delivery of the cell suspension to the empty tissue growth compartment, entrapped air is allowed to exit through port 542 . In a similar fashion,
External acellular compartment 538 is filled with media via port 548 or port 550 and entrapped air can escape via port 544 .

Differentiation bioreactor designs allow direct perfusion of tissue constructs through media delivery to port 546 or indirect supply of media to the surrounding cell-free compartment 538 via port 548 . Typically, ports 542 and 544 are closed during perfusion, with port 550 serving as the media outlet. However, various alternative media supply scenarios are possible based on specific tissue requirements. An important aspect of the medium perfusion strategy is that the permeable membrane 326 forming part of the tissue growth compartment allows fresh culture medium to permeate the tissue growth compartment without any loss of cells from the scaffold. . moreover,
Nutrients are provided to cells from essentially all directions without restriction from any permeable bioreactor walls.

FIG. 22 illustrates a further embodiment of a liquid flow schematic in which the bioreactor of FIGS. 19-21 may be used. There is a tissue digestion chamber 322 that accommodates tissue biopsies.
There is a growth chamber 300 configured to receive cells from the digestion chamber 322 and to allow seeding of growth substrates. Bubble trap 560 removes air bubbles from the input line to the growth chamber, thus preventing these bubbles from entering growth chamber 300 and potentially damaging the localized cell population. Reservoir 562 is adapted to receive expanded cell numbers from growth chamber 300 and to cross-flow filtration module 5 .
It exists to serve as a temporary holding vessel during cell washing and cell concentration procedures performed with the aid of 64. There is also a differentiation and/or tissue formation chamber 306 configured to receive cells from reservoir 562 after washing and concentration steps,
This allows seeding of the implantable scaffold 312 .

Tissue engineering reagents (i.e., media, enzymes, solutions, wash solutions, etc.) are stored in liquid reservoirs 208
a to 208e. Waste products are collected in liquid reservoir 208f. Liquid flow through the system is directed by operation of liquid pumps 122a and 122b, flow control valves 214a-214v, according to control inputs from a central microprocessor. Without compromising the sterility of the system, air filters 566a-566c
Air can be moved into or out of the system as needed during operation. moreover,
In-line gas exchange membranes (not shown) may be used at various locations within the liquid flow path to facilitate control of dissolved gases in the culture medium.

In operation, tissue biopsy 320 is inserted into tissue digestion chamber 322 . Digestion medium containing enzymes is pumped from liquid reservoir 208 into tissue digestion chamber 322 to initiate the digestion process. The digestion medium may be continuously or periodically recirculated by pump 122a to maintain released cells in suspension and maximize reagent exposure to the biopsy. By introducing growth culture medium from one liquid reservoir 208 to the top of the digestion chamber 322, the cell suspension is moved into the growth chamber 300 while the enzyme solution is
It is diluted to a tolerable concentration for cell growth in growth chamber 300 . Movement of partially digested tissue out of the digestion chamber is impeded by port filter 316b, which is sized to allow passage of dissociated cells and retention of cell aggregates. Cells generated from the biopsy digestion process are isolated from valves 214h, 214J, 214I.
and evenly distributed throughout the growth chamber 300, either by recirculation of the cell suspension via actuation of pump 122a, or by the automatic application of a gentle osmotic of the growth bioreactor.

After a rest period to allow cells to adhere to the growth substrate, this growth medium is periodically or continuously replaced with fresh growth medium from one of the liquid reservoirs 208 . During the medium exchange step, the supply of fresh medium from liquid reservoir system 208 is switched off by valve 214i.
is equilibrated by the removal of liquid to waste reservoir 208f via .

Once the cell culture approach reaches confluence, the medium in growth chamber 300
Drains into waste reservoir 208f. In this process, liquid removal from the growth chamber is balanced by the incoming sterile air delivered through sterile filter 566a or by the incoming PBS wash solution from one of the liquid reservoirs 208. .

Cells are subsequently subjected to automated procedures, such as delivery of enzymes (e.g., trypsin) and timed recirculation of cell suspensions, or timed application of impact or agitation to the bioreactor via an impact drive. Released from the growth substrate. Cell growth chamber 306
Cell suspension is transferred from growth chamber 300 to reservoir 562 for removal of enzymes and collection of cells in a relatively small volume of medium for subsequent transfer to. The cell suspension is then continuously recirculated through cross-flow filtration module 564 via valves 214m, 214j, 214q and pump 122a. A membrane in cross-flow filtration module 564 prevents cell loss, but a certain percentage of media (permeate) is allowed to drain to waste reservoir 2 via valve 214o.
Allow to be removed to 08f. As a result, the removal of permeate is
A decrease in suspension volume and/or dilution of any enzyme present is obtained under conditions compensated by a supply of fresh medium from one of the. This continuous flow reduces the possibility of cells being entrapped within the membrane of the cross-flow module 564 .

Reservoir 562 via valves 214m, 214j, 214p and pump 122a
Cell seeding onto the implantable differentiation scaffold 312 is achieved by the migration of washed cells from the top surface of the scaffold. Cell loss from the scaffold is minimized through the optional use of a scaffold membrane or mesh 326 . After cell seeding, fresh differentiation medium was added to pump 1
It may be introduced into the differentiation and/or tissue formation chamber 306 by act 22b.
This differentiation medium is periodically or continuously replaced with fresh differentiation medium from the reservoir system. During the medium exchange process, the supply of fresh medium from one of the liquid reservoirs 208 is balanced by the removal of liquid to waste reservoir 208f via valve 214f. During the medium exchange step, fluid within the differentiation and/or tissue formation chamber is pumped through pump 122b, valve 214t, and valve 214r for perfusion through the tissue construct or valve 214s for delivery outside of scaffold membrane 326. is continuously or periodically recirculated under the control of either The secondary liquid delivery channels outside the scaffold membrane are located away from the area of the implantable scaffold that is seeded with cells, thereby potentially impairing the formation of cell aggregates purely damage stress. is minimal. As with the previous embodiment of the liquid flow schematic, the environmental conditions within the differentiation and/or tissue formation chamber are monitored and controlled for the period necessary for successful formation of the tissue construct, at which point the bioreactor's differentiation and /or the tissue formation chamber is opened and the construct retrieved for subsequent clinical or research use.

Figure 23 illustrates an embodiment of the invention in which the tissue engineering module described herein comprises three bioreactors. FIG. 23 shows FIG. 19 with an internal tissue digestion chamber 322
20 with growth chamber 300 and differentiation bioreactor with growth chamber 306 of FIG. These bioreactors are operatively connected onto the tissue engineering module to provide automated processes involved in tissue digestion, cell growth, cell differentiation and tissue formation procedures.

It will be appreciated by those skilled in the art that an automated tissue engineering system may comprise one or more bioreactors as supported in a housing, either by structural supports or by equivalent means. Where two or more bioreactors are provided, the bioreactors may be operably connected, or may be independently operable and/or operable simultaneously. Further, each bioreactor may have different internal chambers or the same type of chambers. In a further embodiment, the chambers and/or bioreactors are operably connected to provide fluid, cell and/or tissue exchange between the chambers and/or bioreactors.

The automated tissue engineering system of the present invention is easy to prepare for use. The following procedures are representative examples for the preparation of cartilage implants based on the use of the tissue engineering system of the present invention for repair of focal defects in articular cartilage. For this application, steps of tissue digestion, cell proliferation and cell differentiation and/or tissue formation are required. The three stages of the tissue engineering process may be accomplished by a single bioreactor with multiple chambers, or by three separate and distinct bioreactors, as shown in FIGS.

Prior to beginning the tissue engineering procedure, fill reservoirs 208a-208e in the tissue engineering module via reservoir injection port 212 with the following reagent compositions. Reagent A is utilized for digestion of chondrocytes derived from small human articular cartilage biopsies. Reagents B, D and E are utilized for cell growth. Reagent C is utilized for differentiation and tissue construct formation.
Reagent A - Digestion Medium: DMEM/F - 12.5% FCS or autologous serum, 1 μg/
ml insulin, 50 μg/ml ascorbic acid, 100 IU/100 μg/ml
Pen/Strep, 2.5% Hepes Buffer, 0.1%
(1 mg/ml) pronase and 0.025% (0.25 mg/ml) collagenase,
pH 7.4.
Reagent B - Growth medium: DMEM/F-12, 10% FCS or autologous serum 10μ
g/ml 10 μg/ml ascorbic acid, 100 IU/100 μg/ml Pen/
Strep, 2.5% Hepes Buffer, pH 7.4.
Reagent C--differentiation medium: DMEM/F-12, 10% FCS or autologous serum, 1 μg
/ml insulin, 50 μg/ml ascorbic acid, 100 IU/100 μg/ml
Pen/Strep, 2.5% Hepes Buffer, pH 7
. 4.
・ Reagent D-PBS Wash solution: 137 mM NaCl, 3.7 mM KCl, 8 mM
_Na2HPO4 ^* _2H2O , 1.5 _{mM KH2PO4} in water, pH _7.4 .
・Reagent E-cell release solution: 1× trypsin solution

The above reagents are nominally stable for several weeks when stored at 4° C. on the tissue engineering module within the system enclosure. Enzymes may be stored lyophilized within the tissue engineering module and hydrated at the point of use. This allows custom enzymes to be tailored for specific tissue engineering applications.

Human cartilage biopsies (100-500 mg) are obtained by arthroscopic surgery from the non-weight bearing area of the upper medial femoral condyle. Prior to loading the biopsy into the digestion chamber, the biopsy is weighed and the mass recorded for subsequent data entry into the programming procedure for the base unit.
After mass determination, this biopsy is placed in the digestion chamber and the bioreactor is closed, readying the tissue engineering module to be inserted into the base unit of the tissue engineering system. Once the tissue engineering module is installed, the user interface then programs the CPU of the base unit according to the desired biopsy size and tissue engineering procedure.

At the start of the programmed automatic procedure, pronase/collagenase digestion of the biopsy is initiated by injecting Reagent A into the digestion chamber of the bioreactor through actuation of the necessary liquid valves and operation of the liquid delivery pumps. . Digestion is performed at 37° C. for 16 hours with continuous or intermittent recirculation of Reagent A to keep cells in suspension and maximize reagent exposure to the biopsy. This may be followed by two successive washing steps with Reagent D. Approximately 200,000-500,000 per 100 mg of biopsy tissue at the end of the digestion procedure
Obtain 0 cells.

At this point, a sample of digested cells may be collected via the sampling port to assess cell number and viability. This biological assessment is typically assessed outside the system by post-staining hemocytometers with trypan blue.

Under automatic control of the base unit, the dissociated cells are placed on a growth substrate or substrate present in the growth chamber of the bioreactor to obtain a cell seeding density of 2000 cells/cm ² to 15000 cells/cm ² . Deliver on scaffold. Supply reagent B from a reservoir on the tissue engineering module according to the programmed flow profile for continuous growth towards confluence. The temperature and pH of this medium were monitored and each
Detect deviations from 7° C. and pH 7.4. In addition, the state of cell growth is indirectly assessed by detection of turnover as a function of time (eg pH, _O2 , _CO2 , lactate and glucose consumption). The level of confluence is further aided by visual monitoring via a CCD camera connected to a growth probe built into the growth chamber. Once impending confluence was determined either empirically or by sensor-based monitoring, the cells were washed extensively by two successive washing steps with Reagent D to remove all culture medium. is removed.

Detachment of grown cells from the growth substrate or scaffold is initiated by movement of Reagent E from a reservoir within the tissue engineering module to the growth chamber. This trypsin solution resides in the bioreactor for 5 minutes where the reaction is stopped by the automatic addition of Reagent B containing FCS or autologous serum to inhibit enzyme activity. Liberation of cells from the growth substrate or scaffold is further enhanced by applying low frequency impacts to the bioreactor via an impact drive or recirculating the trypsin solution. Once liberated, cell washing and filtration steps are performed to remove trypsin and concentrate the cell suspension for subsequent transfer to the scaffold present in the bioreactor for differentiation and/or tissue formation.

A bipolar configuration is ideal for this application. Because this provides a layer of cartilage at the articular surface, which, for integration with the subchondral bone, Skelite™
It is bonded to a porous scaffolding layer formed from a bone biomaterial such as. Preparation of bipolar constructs may be accomplished by one of several alternative procedures. A differentiation scaffold may be formed by a pore density gradient, which preferentially traps cells at one end and
Creates areas of high cell density that promote cartilage layer formation. Alternatively, the scaffold may be pre-coated at one end with fibrin gel to prevent cell attachment and cartilage matrix formation in this area. By either approach, cell loss from the scaffold is minimized through the optional use of a surrounding membrane or mesh. Flow rates for cell delivery are low to ensure that liquid shear does not damage the growing cell population. After completion of the cell seeding step, fluid flow through the differentiation and/or tissue formation chamber is stopped to allow the formation of cell aggregates. This is because the formation of cell aggregates is known to be important for successful differentiation. This critical step is followed by perfusion of Reagent C for the period required for tissue formation and maturation,
Nutrients are supplied to cells as needed and waste products are removed. After this culture period, the cells produced an extracellular matrix substantially identical to that of native human articular cartilage. The properties of the tissue formed can be confirmed by independent external biochemical methods such as collagen typing via SDS-PAGE and gene expression. As a final step in the process, a notification is obtained from the tissue engineering system via the user interface that the procedure is complete and the tissue engineering module may be removed to retrieve the implant. The tissue engineering module or detachable version of the bioreactor may be moved to a working room where the bioreactor lid is moved to a sterile area to retrieve the implant for surgical use.

It should be noted that the system of the invention is not limited to any particular type of cell or tissue. For example, skeletal implants can be prepared for use in reconstructing bone defects. In this application, bone marrow may be used as a source of primary and/or progenitor cells required for the tissue engineering process. Therefore, there is no need to perform tissue digestion. Thus, the bioreactor may be of a type that supports only proliferation and differentiation. Depending on the available cell population and the required size of the implant, even proliferation may not be necessary. In this case, the bioreactor configuration may be directed to a single stage of cell differentiation and ongoing tissue formation. The final tissue construct is composed of an implantable scaffold, which may be composed of a bone biomaterial such as Skelite™, which lines the open pores of the scaffold. and have active osteocytes that actively anchor new mineralized matrix (bone-like). Such implants are rapidly taken up at the implant site, thereby accelerating the healing process.

As a further example of the flexibility of this system, tissue-engineered blood vessels may be prepared using culture-expanded endothelial cells seeded onto tubular-shaped flexible scaffolds at the terminal differentiation stage. good.

The integrated tissue engineering system of the present invention has several advantages over prior art methods and systems. Specifically, the turnkey operation of this device enables complex tissue engineering procedures to be performed in the clinic under automated control, centralizing cells for biological processing. There is no need to move to the designated facility. The system is simple to use and obviates existing time-consuming and expensive human tissue culture procedures that often result in implant contamination and failure. The combination of this tissue engineering module and related sub-systems allows custom cell or tissue types to be cultured, which can be manufactured from any suitable biocompatible and sterilization-tolerant material. can be done. The entire tissue engineering module or certain components thereof are replaceable and can be considered disposable. This tissue engineering module
It may be provided in a single-use, sterile packaging that facilitates setup and operation of the system in a clinical setting.

Those skilled in the art will appreciate that the tissue engineering modules and devices of the present invention can be manufactured in a variety of sizes, shapes and orientations. The device can be manufactured to incorporate a single tissue engineering module or multiple modules in a vertical or horizontal format. Accordingly, the subassembly can be made to correspond to the spatial modalities selected for the tissue engineering device. Therefore, different types of tissue engineering are
They may be performed simultaneously in a single device, where each tissue engineering procedure is automatically monitored and controlled on an individual basis. It is also within the scope of the invention to have multiple automated tissue engineering systems networked together operating under remote computer control.

While the preferred embodiment of the invention has been described in detail herein, it will be appreciated by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. understood.

Claims (138)

An automated tissue engineering system comprising:
a housing;
at least one bioreactor supported by said housing, said bioreactor facilitating physiological cell function and/or production of one or more tissue constructs from a cell and/or tissue source;
a liquid containment system supported by the housing and in fluid communication with the bioreactor;
one or more sensors coupled with one or more of said housings, bioreactors or liquid containment systems to monitor parameters associated with said physiological cell function and/or production of tissue constructs;
a microprocessor coupled to one or more of the sensors;
A tissue engineering system comprising: The bioreactor is
a bioreactor housing;
one or more inlet ports and one or more outlet ports for the flow of media;
at least one chamber configured within said bioreactor housing for receiving various cells and/or tissues;
2. The system of claim 1, comprising: 3. The system of claim 2, wherein said bioreactor housing comprises a lid, and wherein said one or more inlet and outlet ports are provided within said bioreactor housing and/or said lid. said chambers are tissue digestion chambers, culture and/or proliferation chambers, differentiation and/or
or a tissue formation chamber, and combinations thereof. 5. The system of claims 2, 3 or 4, wherein two or more chambers are provided in operable connection within said bioreactor. 6. The system of claim 2, 3, 4 or 5, wherein said chamber contains one or more substrates and/or scaffolds. 7. The system of claim 6, wherein said scaffold is selected from the group consisting of porous scaffolds, porous scaffolds with gradient porosity, porous reticulated scaffolds, fibrous scaffolds, membrane-enhanced scaffolds, and combinations thereof. 7. The scaffold is selected from the group consisting of natural bioceramics, synthetic bioceramics, natural biopolymers, synthetic biopolymers, implantable bioceramics, implantable biopolymers and mixtures thereof. system. Said substance is an isolated bioresorbable biomaterial compound, said compound comprising calcium, oxygen and phosphorus, wherein a fraction of at least one of these elements is about 0.1 9. The system of claim 8 substituted with elements having an ionic radius of ~0.6 Angstroms. 7. The system of claim 6, wherein the substrate is a suspension containing microcarriers. The system of any one of claims 2-10, wherein the chamber comprises multiple zones. 12. The system of claim 11, wherein each of said plurality of zones can conform to a scaffold and/or substrate. 13. The system of any one of claims 1-12, wherein two or more bioreactors are operably linked. 14. The system of any one of claims 5-13, wherein said two or more chambers and/or bioreactors are operable independently and/or cooperatively. 15. Any one of claims 5 to 14, wherein said chambers and/or bioreactors are operatively connected to provide fluid, cell and/or tissue exchange between said chambers and/or said bioreactors. system of terms. said chambers and/or bioreactors are connected via passageways, tubing, connectors, valves, pumps, filters, liquid access ports, in-line gas exchange membranes, in-line sensors, and/or voids in the separation walls; 16. The system of claim 15. 3. The system of Claim 2, wherein said bioreactor further comprises a sampling port operably connected to one or more of said chambers. 18. The system of any one of claims 1-17, wherein the bioreactor is removably connected to the liquid containment system via the inlet port and the outlet port. 19. The system of any one of claims 1-18, wherein the bioreactor and the liquid containment system are operatively attached to a structural support secured to the housing. 19. The system of any one of claims 1-18, wherein the bioreactor and the liquid containment system are operatively attached to a structural support that is removably connected within the housing. said liquid containment system further comprising one or more flow control valves and one or more pump units operatively connected with said bioreactor and liquid containment system;
System according to any one of claims 1-20. wherein said system further comprises an additional microprocessor and/or logic controller, said microprocessor and/or logic controller facilitating, monitoring and controlling different aspects of tissue engineering in said at least one bioreactor; 22. The system of any one of claims 1-21, programmed to . The bioreactor is capable of tissue biopsy storage, tissue biopsy digestion, cell sorting, cell washing, cell concentration, cell seeding, cell growth, cell differentiation, cell storage, cell transport, tissue formation, implant formation, transplantation. 23. Providing an environment for physiological cell function and/or production of tissue constructs selected from the group consisting of tissue storage, transplantable tissue transport and combinations thereof. system of terms. 24. The system of claim 23, wherein said cells and tissues are selected from bone, cartilage, related bone and cartilage precursor cells, and combinations thereof. embryonic stem cells, adult stem cells derived from bone, bone marrow or blood, wherein said cells comprise stem cells;
24. The system of claim 23, wherein the system is selected from the group consisting of osteoblasts, pre-osteoblasts, chondrocytes, pre-chondrocytes, nucleus pulposus cells, skeletal cells, skeletal progenitor cells, and combinations thereof. The system of any one of claims 1-25, wherein the system is portable. An automated tissue engineering system comprising:
a housing;
at least one tissue engineering module removably connected within said housing, said tissue engineering module being a support structure holding at least one bioreactor, said bioreactor being adapted for cell culture and/or or a support structure that facilitates tissue engineering function, a fluid containment system in fluid communication with said bacteriophage;
and one or more sensors for monitoring parameters associated with said cell culture and/or tissue engineering functions;
a microprocessor disposed in the housing and coupled to the tissue engineering module, the microprocessor controlling operation of the tissue engineering module;
A tissue engineering system comprising: The bioreactor is
a bioreactor housing having one or more inlet ports and one or more outlet ports for the flow of media;
at least one chamber defined within said bioreactor housing for receiving cells and/or tissue and for facilitating said cell culture and tissue engineering functions;
28. The tissue engineering system of claim 27, comprising: said chambers are tissue digestion chambers, culture and/or proliferation chambers, differentiation and/or
or a tissue formation chamber, and combinations thereof. 30. The tissue engineering system of claim 29, wherein said chamber contains one or more substrates and/or scaffolds. two or more chambers are provided in operable connection within said bioreactor;
31. The tissue engineering system of claim 28, 29 or 30. 32. The tissue engineering system of any one of claims 27-31, wherein two or more bioreactors are operatively connected. 33. The tissue engineering system of claim 31 or 32, wherein said two or more chambers and/or bioreactors are independently operable and/or cooperatively operable. 28. The tissue engineering system of Claim 27, wherein said bioreactor is removable. 35. Any one of claims 27 to 34, wherein said chambers and/or bioreactors are operably connected to provide fluid, cell and/or tissue exchange between said chambers and/or said bioreactors. Section Tissue Engineering System. 31. The tissue engineering system of claim 30, wherein said scaffold is selected from the group consisting of porous scaffolds, porous scaffolds with gradient porosity, porous reticulated scaffolds, fibrous scaffolds, membrane-enhanced scaffolds, and combinations thereof. said chamber comprises multiple zones to contain multiple substrates and/or scaffolds;
37. The tissue engineering system of claim 36. The bioreactor is capable of tissue biopsy storage, tissue biopsy digestion, cell sorting, cell washing, cell concentration, cell seeding, cell growth, cell differentiation, cell storage, cell transport, tissue formation, implant formation, transplantation. 2. Providing an environment for cell culture and/or tissue engineering functions selected from the group consisting of tissue storage, transplantable tissue transport and combinations thereof.
38. The tissue engineering system of any one of Clauses 7-37. The tissue engineering module has a flow control valve connected to the fluid containment system, the tissue engineering module operably engaging one or more valve actuators, a pump unit and a connector provided within the housing. 39. The tissue engineering system of claim 38. 40. The tissue engineering system of Claim 39, wherein said connector provides electrical connection between said tissue engineering module and said microprocessor via an electrical backplane provided within said housing. 28. The tissue engineering system of claim 27, wherein said fluid containment system comprises a plurality of flexible reservoirs connected by flexible tubing to supply and withdraw fluid to and from said bioreactor. The housing comprises one or more environmental sensors and environmental control units to maintain environmental conditions within one or more of the housing, tissue engineering module, bioreactor, fluid containment system, and/or the flexible reservoir. 42. The tissue engineering system of any one of claims 27-41, comprising: 43. The tissue engineering system of Claim 42, wherein said environmental conditions within said bioreactor are maintained independently of said fluid containment system. 43. The tissue engineering system of claim 42, wherein the environmental conditions within the one or more flexible reservoirs are maintained independently within the fluid containment system. 44. The tissue engineering system of claim 43, wherein liquid entry into said bioreactor is provided at a temperature that essentially corresponds to the liquid temperature within said bioreactor. The environmental conditions within the bioreactor are maintained at a temperature suitable for cell culture and/or tissue engineering functions, and the environmental conditions within the liquid containment system or selected flexible reservoir are maintained at the cell culture and/or tissue engineering function. 44. The tissue engineering system of claim 43, wherein the tissue engineering system is maintained at a temperature below that appropriate for tissue engineering function. 28. The tissue engineering system of claim 27, wherein said housing has at least one insertion slot for insertion of said tissue engineering module through at least one set of guides for receiving said support structure. 48. The tissue engineering system of claim 47, wherein said insertion slot and said guide are positioned vertically or horizontally with respect to said housing. 49. The tissue engineering system of claims 46 or 48, wherein said insertion slot has a movable door and locking mechanism. 49. The system of any one of claims 27-49, further comprising a user interface cooperating with the microprocessor, the user interface providing input of user input and/or output of system status to the microprocessor. tissue engineering system. 51. The tissue engineering system of claim 50, wherein said user interface is selected from displays, touch screens, keypads, computers, computer networks, computer data media devices and combinations thereof. 52. The tissue engineering system of claim 50 or 51, further comprising a data system allowing input, output, recording, transfer and storage of information. 53. The tissue engineering system of Claim 52, further comprising a computer and communication circuitry. 28. The tissue engineering system of claim 27, wherein said microprocessor controls independent operation of multiple tissue engineering modules within said housing. 28. The tissue engineering system of Claim 27, wherein said microprocessor performs quality control assessments of said cell culture and tissue engineering functions. 56. The tissue engineering system of Claim 55, wherein said quality control evaluation assessment is presented as a quality control record via said user interface. 28. Said microprocessor forms part of said tissue engineering module.
tissue engineering system. 28. The tissue engineering system of claim 27, wherein said tissue engineering module further comprises one or more microprocessors operatively coupled with said microprocessor located in said housing. A portable and sterilizable tissue engineering module, said module comprising:
a structural support holding at least one bioreactor, said bioreactor facilitating cell culture and tissue engineering functions;
a liquid containment system in fluid communication with the bioreactor;
one or more sensors for monitoring parameters associated with said cell culture and tissue engineering functions;
a tissue engineering module. The bioreactor is
a bioreactor housing having one or more inlet ports and one or more outlet ports for the flow of media;
60. The tissue engineering module of claim 59, comprising at least one chamber defined within said bioreactor housing for receiving cells and/or tissue and for facilitating cell culture and tissue engineering functions. said chambers are tissue digestion chambers, culture and/or proliferation chambers, differentiation and/or
or a tissue formation chamber, and combinations thereof. 61. The tissue engineering module of claim 60, wherein said chamber contains one or more substrates and/or scaffolds. two or more chambers are provided in operable connection within said bioreactor;
61. The tissue engineering module of claim 60. 64. The tissue engineering module of any one of claims 59-63, wherein two or more bioreactors are operably connected. 65. The tissue engineering module of claim 63 or 64, wherein said two or more chambers and/or bioreactors are independently operable and/or cooperatively operable. The module comprises a first bioreactor, a second bioreactor and a third bioreactor, wherein the first bioreactor comprises a tissue digestion chamber and the second bioreactor is a culture and/or growth chamber. and wherein said third bioreactor comprises a differentiation and/or tissue formation chamber, wherein said first, second and third bioreactors are operably connected module. 67. Any one of claims 63 to 66, wherein said chambers and/or bioreactors are operatively connected to provide fluid, cell and/or tissue exchange between said chambers and/or said bioreactors. Section Tissue Engineering Module. 63. The tissue engineering module of claim 62, wherein said scaffold is selected from the group consisting of porous scaffolds, porous scaffolds with gradient porosity, porous reticulated scaffolds, fibrous scaffolds, membrane-enhanced scaffolds, and combinations thereof. said chamber comprises multiple zones to contain multiple substrates and/or scaffolds;
63. The tissue engineering module of claim 60 or 62. The bioreactor is capable of tissue biopsy storage, tissue biopsy digestion, cell sorting, cell washing, cell concentration, cell seeding, cell growth, cell differentiation, cell storage, cell transport, tissue formation, implant formation, transplantation. 59. Providing an environment for cell culture and/or tissue engineering functions selected from the group consisting of tissue storage, transplantable tissue transport and combinations thereof.
The tissue engineering module of any one of Clauses 1-69. 60. The tissue engineering module of Claim 59, wherein said fluid containment system comprises a plurality of flexible reservoirs connected by flexible tubing to supply and withdraw fluid to and from said bioreactor. 72. The tissue engineering module of claim 71, wherein said flexible reservoirs include various configurations and sizes. 72. The tissue engineering module of claim 71, wherein said flexible reservoir and/or tubing is provided with a fluid access port for filling or removing material from said fluid containment system. a plurality of liquid flow control valves in operable communication with a liquid containment system for controlling the flow of liquid using the liquid containment system and the bioreactor;
60. The tissue engineering module of claim 59. 75. The tissue engineering module of claim 74, wherein said fluid flow control valve is opened and closed by a valve actuator. 1, wherein said tissue engineering module is provided within a housing of an automated tissue engineering device;
60. The tissue engineering module of claim 59, operatively engageable with one or more valve actuators, pump units and connectors. 77. The tissue engineering module of claim 76, wherein said module further comprises one or more pump units in communication with said fluid containment system for pumping fluid through said fluid containment system. A liquid flow plate is integrally mounted or provided with the structural support, the liquid flow plate directing liquid flow into and between the liquid containment system and the bioreactor. Claim 59 operably connected to the control valve
Tissue Engineering Module. 79. The tissue engineering module of any one of claims 59-78, further comprising a heating and mixing chamber for heating and mixing liquids that the tissue engineering module flows to the bioreactor. One or more gas exchange membranes are provided in or between said liquid containment system and/or bioreactor, said gas exchange membranes allowing gaseous products to flow into or out of said bioreactor. 60. The tissue engineering module of claim 59, capable of locomotion or lodging in said bioreactor. 60. The tissue engineering module of Claim 59, wherein said module further comprises a thermoelectric element operably connected to said bioreactor. 60. The tissue engineering module of Claim 59, wherein said module further comprises one or more microprocessors operatively connected with said sensors. 81. Said module further comprises a printed circuit board to provide an electrical interface for one or more of said sensors, microprocessors or thermoelectric elements.
or 82 tissue engineering modules. said module further comprising one or more access ports, said access ports operably coupled to said bioreactor and/or fluid containment system, said access ports being adapted to store cells, liquids, cell and tissue culture media; 60. The tissue engineering module of claim 59, which provides aseptic loading or removal of growth factors, pharmaceutical agents, quality control reagents, quality control samples and other substances. 85. The tissue engineering module of claim 84, wherein said access port is operably connected to a syringe manifold integrated with said structural support. 86. The tissue engineering module of claim 85, wherein said syringe manifold comprises at least one access port connected to a syringe. 60. The tissue engineering module of Claim 59, wherein said bioreactor is rotatably mounted to said structural support. 60. The tissue engineering module of Claim 59, wherein said bioreactor is integrated and attached to said structural support. 60. The tissue engineering module of Claim 59, wherein said bioreactor is detachable from said structural support. Claims 59-89, wherein a camera is provided for visual inspection within said bioreactor
The tissue engineering module of any one of . 60. The tissue engineering module of Claim 59, wherein said module further comprises an identification element selected from the group consisting of barcodes, magnetic strips, ID tags, electronic memories and combinations thereof. 60. The tissue engineering module of Claim 59, wherein said module is sterilizable prior to use and disposable after use. 60. The tissue engineering module of claim 59, wherein said module is suitable for sorting and/or transporting cells and tissues at different temperatures. 93. The tissue engineering module of claim 92, wherein said module is provided within sterile packaging. A bioreactor for facilitating or supporting cellular function and/or production of tissue constructs, said bioreactor comprising:
a bioreactor housing;
one or more inlet ports and one or more outlet ports for the flow of media;
at least one chamber defined within said bioreactor housing to facilitate and support cell function and/or production of one or more tissue constructs from a cell and/or tissue source;
and one or more sensors for monitoring parameters associated with said cell function and/or tissue construct production within said at least one chamber. 96. The bioreactor of claim 95, wherein said bioreactor housing comprises a lid, said lid being selected from removable lids and lids integral with said bioreactor housing. said chambers are tissue digestion chambers, culture and/or proliferation chambers, differentiation and/or
or a tissue formation chamber, and combinations thereof. 99. The bioreactor of Claim 98, wherein said chamber supports one or more substrates and/or scaffolds. 97. Claim 97 or 9, wherein said bioreactor comprises a single culture and/or growth chamber having a culture and/or growth scaffold and/or substrate supported therein.
8 bioreactors. said bioreactor comprising a culture and/or growth chamber connected to a downstream differentiation and/or tissue formation chamber, said culture and/or growth chamber having a growth scaffold and/or a substrate supported thereon; 99. The bioreactor of claim 97 or 98, wherein said differentiation and/or tissue formation chamber has an implantable differentiation scaffold supported therein. said connecting passage is provided between said culture and/or proliferation chamber and said differentiation and/or tissue formation chamber, said connecting passage facilitating passage of released cells from said proliferation scaffold and/or substrate to said differentiation scaffold; 99. The bioreactor of claim 97 or 98, which facilitates movement. 102. The bioreactor of claim 101, wherein said bioreactor further comprises a digestion chamber for digestion of tissue biopsy material, said digestion chamber upstream of said culture and/or growth chamber. 10. One or more filters are provided at a location selected from upstream of the proliferation scaffold, upstream of the differentiation scaffold, upstream of the outlet port and combinations thereof.
0, 101 or 102 bioreactors. Claim 98-1, wherein said scaffold is selected from the group consisting of a porous scaffold, a porous scaffold with gradient porosity, a porous reticulated scaffold, a fibrous scaffold, a membrane-enhanced scaffold, and combinations thereof.
03. The bioreactor of any one of clause 03. 4. The scaffold is selected from the group consisting of natural bioceramics, synthetic bioceramics, natural biopolymers, synthetic biopolymers, implantable bioceramics, and implantable biopolymers and mixtures thereof. 104 bioreactors. 99. The bioreactor of claim 98, wherein said growth substrate is a suspension containing microcarriers. 102. The bioreactor of claim 101, wherein said differentiation and/or tissue formation chamber comprises multiple zones to contain multiple differentiation scaffolds and/or substrates. 96. The bioreactor of Claim 95, wherein said bioreactor has a sampling port operably connected to one or more of said chambers. 96. The bioreactor of Claim 95, wherein a gas exchange membrane forms part of said chamber. 96. The bioreactor of Claim 95, wherein said bioreactor is removably connected to a liquid containment system via said inlet port and said outlet port. 96. The bioreactor of claim 95, wherein a mixing diaphragm forms part of said chamber and is operably connected to a mixing actuator and/or mixing drive. 112. The bioreactor of claim 111, wherein said mixing diaphragm is incorporated within said bioreactor. 96. The bioreactor of claim 95, wherein said bioreactor is operably connected to an impact actuator and/or impact drive. 96. The bioreactor of claim 95, wherein said chamber further supports physiological stimulation of lodging cells and/or tissue. 115. The bioreactor of claim 114, wherein said stimulation is performed by a microfilling diaphragm operatively connected with a microfilling actuator and/or a microfilling drive. Claim 1, wherein said microfill diaphragm is incorporated within said bioreactor.
15 bioreactors. 115. The bioreactor of Claim 114, wherein said stimulation is performed by providing an electric field. The bioreactor is operatively connected within a tissue engineering module that includes a fluid containment system secured to a structural support and in fluid communication with the bioreactor, the fluid containment system communicating with the bioreactor. 118. The bioreactor of any one of claims 95-117, comprising a plurality of flexible reservoirs connected by flexible tubing for supplying and withdrawing liquid from said bioreactor. 119. The bioreactor of claim 118, wherein said tissue engineering module is connected within a housing of an automated tissue engineering system, said system comprising a microprocessor for operating said module, said microprocessor controlling the function of said module. . 120. The bioreactor of any one of claims 95-119, wherein said bioreactor further comprises an optical probe. 95. The bioreactor of claim 95, wherein said bioreactor can be used to store or transport cells, tissues and/or implants under sterile conditions. Said bioreactor is capable of: storage of tissue biopsies, digestion of tissue biopsies, cell sorting, cell washing, cell enrichment, cell seeding, cell proliferation, cell differentiation, cell storage, cell transport, tissue formation, implant formation, 122. The bioreactor of any one of claims 95-121, which provides an environment for one or more selected from the group consisting of storage of implantable tissue, transport of implantable tissue. 123. The bioreactor of claim 122, wherein said cells and tissues are selected from bone, cartilage, related bone and cartilage precursor cells, and combinations thereof. embryonic stem cells, adult stem cells derived from bone, bone marrow or blood, wherein said cells comprise stem cells;
123. The bioreactor of claim 122, wherein the bioreactor is selected from the group consisting of osteoblasts, pre-osteoblasts, chondrocytes, nucleus pulposus cells, skeletal cells, pre-chondrocytes, skeletal progenitor cells, and combinations thereof. 125. The bioreactor of claims 123 or 124, wherein said cells or said tissue are autologous, allogeneic, or xenogeneic to the recipient of the implant. 96. The bioreactor of claim 95, wherein said bioreactor is sterilizable prior to use and disposable after use. 127. The bioreactor of claim 126, wherein said bioreactor is provided within sterile packaging. A method for automated digestion of a tissue biopsy, comprising:
filling a tissue biopsy into a bioreactor connected with a media reservoir and a flow system, the bioreactor being adapted to detect physiological conditions within the bioreactor for assessment by a microprocessor; having one or more sensors;
A method comprising: providing tissue-digesting enzymes within said bioreactor; and monitoring and maintaining digestion conditions within said bioreactor for a time sufficient for a desired level of tissue digestion. A method for automated proliferation of cells, comprising:
Seeding cells onto a growth substrate or scaffold supported within a bioreactor connected to a media reservoir and flow system, said bioreactor being adapted for microprocessor-assisted assessment of physiological conditions within said bioreactor. monitoring and maintaining appropriate culture conditions within said bioreactor for a time sufficient for a desired level of cell growth. . A method for automated differentiation of cells, comprising:
Seeding cells onto a differentiation substrate or scaffold supported within a bioreactor connected to a media reservoir and flow system, said bioreactor being adapted for microprocessor-assisted assessment of physiological conditions within said bioreactor. monitoring and maintaining appropriate culture conditions within said bioreactor for a time sufficient for a desired level of cell differentiation. . A method for the generation of a tissue construct, comprising:
Seeding cells onto a scaffold supported within a bioreactor, said bioreactor being connected to a media reservoir and a flow system, said bioreactor communicating with a microprocessor to physiological conditions within said bioreactor. 1 for detecting
monitoring and maintaining appropriate culture conditions within said bioreactor, having one or more sensors, and for sufficient time for said cells to express an extracellular matrix that provides a structural support for tissue building. A method comprising the step of 1. An automated method for digesting a tissue biopsy to provide primary cells, including progenitor cells, and for further proliferating and differentiating the cells to enable formation of a tissue implant, comprising:
filling a tissue biopsy into a bioreactor connected with a media reservoir and a flow system, the bioreactor being adapted to detect physiological conditions within the bioreactor for assessment by a microprocessor; having one or more sensors;
providing tissue digestive enzymes;
monitoring and maintaining appropriate digestion conditions within said bioreactor for a time sufficient to obtain dissociated cells;
- seeding said dissociated cells on a growth substrate or scaffold supported within a bioreactor connected to a medium reservoir and flow system, said bioreactor being connected to said bio for assessment by a microprocessor; having one or more sensors for detecting physiological conditions within the reactor;
monitoring and maintaining appropriate culture conditions within said bioreactor for a time sufficient to obtain a desired level of cell differentiation and proliferation;
releasing the expanded cells from the growth substrate or scaffold;
seeding the expanded cells onto a differentiation substrate or scaffold supported within a bioreactor connected to a media reservoir and a flow system, the bioreactor being controlled within the bioreactor for assessment by a microprocessor; and monitoring and maintaining appropriate culture conditions within said bioreactor for a time sufficient to obtain a tissue implant. Method. A method for providing a skeletal implant, comprising:
seeding osteogenic and/or osteoprogenitor cells in a bone biomaterial porous scaffold supported within a bioreactor connected to a media reservoir and flow system, said bioreactor comprising a micro having one or more sensors for detecting physiological conditions within said bioreactor for assessment by a processor;
and for a period of time sufficient to allow the osteogenic and/or osteoprogenitor cells to proliferate and/or differentiate throughout the scaffold to provide a tissue implant for orthopedic applications. A method comprising monitoring and maintaining appropriate conditions within a bioreactor. A method for providing a cartilage implant, comprising:
seeding chondrogenic cells and/or chondroprogenitor cells in a biomaterial porous scaffold supported within a bioreactor connected to a media reservoir and a flow system, said bioreactor being assessed by a microprocessor; and said chondrogenic cells and/or throughout said scaffold to provide a cartilage implant.
or monitoring and maintaining appropriate conditions within said bioreactor for a time sufficient to allow chondroprogenitor cells to proliferate and/or differentiate. A method for providing an implant for regenerating the inner nucleus of an intervertebral disc, comprising:
seeding nucleus pulposus cells within a porous scaffold of a biocompatible scaffold supported within a bioreactor connected to a media reservoir and a flow system, said bioreactor communicating with said microprocessor to said having one or more sensors for detecting physiological conditions within the bioreactor and allowing proliferation and/or differentiation of said nucleus pulposus cells and expression of extracellular matrix components characteristic of said nucleus pulposus. monitoring and maintaining suitable conditions within said bioreactor for a period of time sufficient to allow A method for the preparation of quality assessment samples for use in clinical tissue engineering, comprising:
Parallel preparation of primary and secondary implants using the system of claim 1 or 27, wherein the primary implant is for implantation and one or more secondary implants infer the capabilities of the primary implant A method that includes preparation for testing purposes. A method for washing cells, comprising:
filling the chamber with a cell suspension containing one or more undesirable chemicals;
said cell suspension from said chamber through a cross-flow filtration module comprising a membrane impermeable to said cells but permeable to said undesired chemicals to provide a washed cell suspension; and collecting said washed cell suspension. A method for cell enrichment, comprising:
filling a chamber with a cell suspension containing excess cell suspension volume; and being impermeable to said cells, but removing and collecting said excess cell suspension volume. continuously recirculating said cell suspension from said chamber through a cross-flow filtration module comprising a membrane that allows

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